SHC enzymes and enzyme variants

Abstract

The present invention relates to squalene hopaene cyclase (SHC) and variants thereof, and their use in the preparation of (-)-ambergris from homofarnesol, the preparation of ambergris oxides from dihomofarnesol, and the like.

Description

Technical Field

[0001] The present invention generally relates to SHC / HAC enzymes and variants thereof. The present invention also relates to various uses of SHC / HAC enzymes and variants thereof, such as the enzymatic conversion of (3E,7E)-homofarnesol (EEH) to (-)-Ambrox or the enzymatic conversion of E,E-bishomofarnesol (BisEEH) to ambrox and its analogs. The present invention also relates to the products of the enzymatic reactions, such as (-)-Ambrox or ambrox produced using SHC / HAC enzymes and variants thereof, and various uses of such products. Background Art

[0002] Squalene Hopene Cyclase (SHC) is a membrane-bound enzyme that acts as a biocatalyst to cyclize the linear triterpenoid squalene to hopene and hopeol.

[0003] A number of wild-type and variant SHC enzymes from various bacteria have been shown to be useful for converting (3E,7E)-homofarnesol to (-)-ambroxol (see, e.g., WO 2016 / 170099; WO 2018 / 157021; Neumann & Simon 1986, Biol Chem Hoppe-Seyler 367, 723-729; JP 2009060799; Seckler & Poralla 1986, Biochem Biophys Act 356-363; Ochs et al. 1990, J Bacteriol 174, 298-302; WO 2010 / 139719; US 8759043; WO 2012 / 066059; Seitz et al. 2012, J Molecular Catalysis B: Enzymatic 84, 72-77; and Seitz et al. 2013, J Molecular Catalysis B: Enzymatic 84, 72-77). The invention relates to a method for preparing (-)-Ambroxol, for example by using novel SHC enzymes or enzyme variants. The invention also relates to a method for cyclizing other substrates, for example to form compounds that can be used in or as fragrances. Summary of the Invention

[0004] In a first aspect, a method for preparing (-)-Ambroxol or a mixture comprising (-)-Ambroxol is provided, the method comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH into (-)-Ambroxol or a mixture comprising (-)-Ambroxol using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has the same sequence as SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,47,48,49,50,51,52,53,54,55,56,5 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 , 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147 , 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295 The amino acid sequences 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have at least 30.0% identity or similarity in amino acid sequence, and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to the following sequences at positions W169, A306, and / or G600 corresponding to SEQ ID NO: 1, 2, or 3, or at positions W169, A306, and / or G600 corresponding to SEQ ID NO: 1, 2, or 3 in the amino acid sequence of the wild-type SHC / HAC enzyme: SEQ ID NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、1 45, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、 216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、 252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0005] In one embodiment, a method for preparing (-)-Ambroxol or a mixture comprising (-)-Ambroxol is provided, the method comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH into (-)-Ambroxol or a mixture comprising (-)-Ambroxol using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 13, 15, 23, 32, and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid alteration relative to SEQ ID NO: 1 at positions corresponding to positions W169, A306 and / or G600 of SEQ ID NO: 1, or at positions corresponding to W169, A306 and / or G600 of SEQ ID NO: 1 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0006] In one embodiment, a method for preparing ambergris oxide or a mixture containing ambergris oxide is provided, the method comprising enzymatically converting (2,E)-bis-hofarnesol (BisEEH) or a mixture of bis-hofarnesol isomers containing BisEEH to ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has the same characteristics as SEQ ID NO. NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,47,48,49,50,51,52,53,54,55,56,57 ,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,9 3, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 2 02, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296 , 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid alteration at a position corresponding to position W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3 in the amino acid sequence of the wild-type SHC / HAC enzyme relative to the following sequence: SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,3 3, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 21 7, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 28 8, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0007] In one embodiment, a method for preparing ambergris oxide or a mixture containing ambergris oxide is provided, the method comprising enzymatically converting (2,E)-bis-hofarnesol (BisEEH) or a mixture of bis-hofarnesol isomers containing BisEEH to ambergris oxide or a mixture containing ambergris oxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO:1, 13, 15, 23, 32, and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid change relative to SEQ ID NO:1 at positions W169, A306, and / or G600 corresponding to SEQ ID NO:1, or at positions W169, A306, and / or G600 corresponding to SEQ ID NO:1 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0008] In one embodiment, a method is provided, wherein:

[0009] - At position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the letter W is replaced by G, A, or V or their functional equivalent. Preferably, the letter W at position 169 is replaced by G.

[0010] - the A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted by V or a functional equivalent thereof, and / or

[0011] - the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with A, V, L, I or M or a functional equivalent thereof.

[0012] In one embodiment, a method is provided, wherein:

[0013] The 'W' in position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'G'.

[0014] In one embodiment, a method is provided, wherein:

[0015] -In SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the 169th bit W is replaced by G, and

[0016] The 'A' in the 306th position of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'V'.

[0017] In one embodiment, a method is provided, wherein:

[0018] -In SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the 169th bit W is replaced by G, and

[0019] - G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with M.

[0020] In one embodiment, a method is provided, wherein:

[0021] - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with G,

[0022] - the A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with V, and

[0023] - the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with M.

[0024] In one embodiment, a method is provided, wherein:

[0025] In SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,1 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,The amino acid at position 168 of 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 is S, or corresponds to the amino acid sequence of SEQ ID NO: NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 1 21, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172 2, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264 The amino acid at position 168 of 4, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 is S.

[0026] In one embodiment, a method as defined above is provided, wherein the amino acid at position 168 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is S (or the amino acid sequence of wild-type SHC corresponding to amino acid position 168 of SEQ ID NO: 1 is S).

[0027] In one embodiment, there is provided a method as defined above, wherein

[0028] - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 1, 4, 5, 6 or 350 and having at least one of the following mutations: W169G, A306V and G600M, or

[0029] - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 7, 8, 9, or 386, and having the following mutations: M132R, A224V, I432T, A557T, R613S, and having at least one of the following mutations: W169G, A306V, and G600M, or

[0030] - The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 10, 11, 12 or 385, and has the following mutations: M132R, A224V, I432T, and at least one of the following mutations: W169G, A306V and G600M.

[0031] In one embodiment, a method is provided wherein W at position 169 is substituted with G.

[0032] In one embodiment, a method is provided, wherein:

[0033] The W at position 169 was replaced by G, and the A at position 169 was replaced by V.

[0034] In one embodiment, a method is provided, wherein:

[0035] The 169th W was replaced by G, and the 600th G was replaced by M.

[0036] In one embodiment, a method is provided, wherein:

[0037] The 169th W was replaced by G, the 306th A was replaced by V, and the 600th G was replaced by M.

[0038] In one embodiment, there is provided a method as defined above, wherein

[0039] - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360 and having at least one of the following mutations: W172G, A311V and G609M (Tel SHC / HAC variant), or

[0040] - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and having the following mutations: W196G, A335V and G629M (Sco SHC / HAC variant), or

[0041] - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and having at least one of the following mutations: W222G, A368V and G667M (Zmo SHC1 variant), or

[0042] - The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has at least one of the following mutations: W177G, A321V and G619M (Zmo SHC2 variant).

[0043] In one embodiment, a method is provided, wherein:

[0044] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359, or 360, and has a W172G mutation.

[0045] In one embodiment, a method is provided, wherein:

[0046] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359, or 360, and has W172G and A311V mutations.

[0047] In one embodiment, a method is provided, wherein:

[0048] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359, or 360, and has W172G and G609M mutations.

[0049] In one embodiment, a method is provided, wherein:

[0050] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359, or 360, and has W172G, A311V, and G609M mutations.

[0051] In one embodiment, a method is provided, wherein:

[0052] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:32, 311, 357 or 358, and has the W196G mutation.

[0053] In one embodiment, a method is provided, wherein:

[0054] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has W196G and A335V mutations.

[0055] In one embodiment, a method is provided, wherein:

[0056] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:32, 311, 357 or 358, and has the W196G and G629M mutations.

[0057] In one embodiment, a method is provided, wherein:

[0058] The SHC / HAC enzyme variants have an amino acid sequence that is at least 70.0% identical to SEQ ID NO:32, 311, 357 or 358, and have the mutations W196G, A335V and G629M.

[0059] In one embodiment, a method is provided, wherein:

[0060] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:13, 312, 353 or 354, and has the W222G mutation.

[0061] In one embodiment, a method is provided, wherein:

[0062] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353, or 354, and has W222G and A368V mutations.

[0063] In one embodiment, a method is provided, wherein:

[0064] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353, or 354, and has W222G and G667M mutations.

[0065] In one embodiment, a method is provided, wherein:

[0066] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353, or 354, and has W222G, A368V, and G667M mutations.

[0067] In one embodiment, a method is provided, wherein:

[0068] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:15, 313, 355 or 356, and has the W177G mutation.

[0069] In one embodiment, a method is provided, wherein:

[0070] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355, or 356, and has W177G and A321V mutations.

[0071] In one embodiment, a method is provided, wherein:

[0072] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355, or 356, and has W177G and G619M mutations.

[0073] In one embodiment, a method is provided, wherein:

[0074] The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355, or 356, and has W177G, A321V, and G619M mutations.

[0075] In one embodiment, there is provided a method as defined above, wherein the SHC / AHC enzyme variant has an amino acid sequence comprising, consisting of, or consisting essentially of the following sequence: SEQ ID NO: NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 305, 306, 304, 302, 311, 312, 313, 353, 354, 355, 356, 357, 358, 359, 360, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 350, 351 or 352.

[0076] In one embodiment, there is provided a method as defined above, wherein the SHC / HAC enzyme variant disclosed herein exhibits:

[0077] - increased substrate specificity for EEH (or for BisEEH) when using homofarnesol or dihomofarnesol substrates,

[0078] - when homofarnesol or dihomofarnesol substrates are used, the product selectivity towards (-)-ambroxol (or ambergris oxide) is increased,

[0079] - When using substrates other than perfarnesol or bisperfarnesol (e.g., ethyl-perfarnesol, hydroxyfarnesol, etc. - see, for example, WO2021 / 110858 and WO2021 / 209482 respectively), the specificity for specific isomers of the substrate increases.

[0080] -Increased productivity, and / or

[0081] - During the first 3 to 6 hours of the reaction (or the first 4, 5, or 6 hours), the degree of conversion of EEH (or BisEEH) and the conversion rate of EEH (or BisEEH) increase.

[0082] The increase is relative to a reference SHC enzyme (e.g., wild-type SHC, e.g., SEQ ID). NO:1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 7 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9 1, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383).

[0083] In one embodiment, a method as defined above is provided, wherein the enzymatic conversion is carried out at a temperature in the range of about 30°C to about 50°C, for example in the range of about 40°C to about 50°C, and / or in the pH range of about 5 to about 6.

[0084] In one embodiment, a method as defined above is provided, wherein the method includes culturing recombinant host cells that produce SHC / HAC enzyme variants.

[0085] In one embodiment, a method as defined above is provided, wherein the recombinant host cell comprises a nucleic acid sequence selected from SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45, or 46.

[0086] In one embodiment, there is provided a process as defined above, wherein the homofarnesol isomer mixture comprising EEH is an EE:EZ isomer mixture, preferably, wherein the weight ratio of the EE:EZ isomer mixture is 80:20.

[0087] In one embodiment, a method as defined above is provided, wherein the weight ratio of the recombinant host cell producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is about 2:1, 1:1 or about 0.5:1 or about 0.1:1, and the homofarnesol isomer mixture comprising EEH is an 80:20 weight ratio EE:EZ isomer mixture.

[0088] In one embodiment, a method is provided, wherein:

[0089] The weight ratio of the recombinant host cells producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is 0.4, and the concentration of EEH is 450 g / l, the mixture being an EE:EZ isomer mixture.

[0090] In one embodiment, a method is provided, wherein:

[0091] The weight ratio of the recombinant host cell that produces the SHC / HAC enzyme variant to EEH or a mixture of high farnesol isomers containing EEH is 1, and the concentration of EEH is 250 g / L, said mixture being an EE:EZ isomer mixture.

[0092] In one embodiment, a method is provided, wherein:

[0093] The weight ratio of the recombinant host cells producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is 1, and the concentration of EEH is 300 g / l, the mixture being an EE:EZ isomer mixture.

[0094] In another aspect, there is provided a variant of an SHC / HAC enzyme as defined herein.

[0095] In a further aspect there is provided (-)-Ambroxol obtained or obtainable by a process as defined herein, said (-)-Ambroxol being in solid form in amorphous or crystalline form.

[0096] On the other hand, the use of (-)-ambroxan, as obtained herein, as part of fragrances or cosmetics or consumer products such as fabric care, toiletries, beauty care, cleaning products, detergent products and / or soap products is provided.

[0097] In another aspect, there is provided a fragrance or cosmetic or consumer product comprising (-)-Ambroxol as obtained herein.

[0098] On the other hand, nucleic acid sequences encoding SHC / HAC enzyme variants as defined herein are provided.

[0099] On the other hand, constructs containing nucleic acid sequences as defined in this paper are provided.

[0100] In another aspect, there is provided a recombinant host cell comprising a nucleic acid sequence or construct as defined herein. Attached Figure Description

[0101] Figure 1: EEH transformation using AacSHC and 215G2SHC P1, P2 and P3 variants. Reactions were performed using 8 g / l EEH (AacSHC wt only 4 g / l EEH), and the OD of the cells was 650nm The value is 10, and individually optimized reaction conditions (T, pH, SDS) are applied.

[0102] Figure 2 Shown are the reaction products of the SHC-catalyzed homofarnesol cyclization using a mixture of E,E-homofarnesol and E,Z-homofarnesol isomers.

[0103] Figure 3The substrate specificity and product selectivity of P1, P2, and P3 SHC variants were utilized. The reaction was performed using 8 g / L EEH, and the cellular OD... 650nm It is 10, applying individually optimized reaction conditions (T, pH, SDS).

[0104] Figure 4 Homofarnesol cyclization using SHC variants. Reactions were performed at 125 g / l EEH and 250 g / l cells, with T, pH and SDS (SDS:cell ratio) conditions defined as optimal for each variant.

[0105] Figure 5 High farnesol biotransformation was performed using SHC variants. Reactions were carried out at 125 g / L EEH and 250 g / L cells, with T, pH, and SDS (SDS: cell ratio) conditions defined as optimal for each variant.

[0106] Figure 6 Substrate specificity and product selectivity in high-farnesol biotransformation using SHC enzyme variants. Reactions were carried out at 125 g / L E, E-high-farnesol and 250 g / L cells, with T, pH, and SDS (SDS: cell ratio) conditions defined as optimal for each SHC enzyme.

[0107] Figure 7 : Bioconversion of homofarnesol using SHC variants. Reactions were performed at 250 g / l EEH and 250 g / l cells, with T, pH and SDS (SDS:cell ratio) conditions defined as optimal for each variant.

[0108] Figure 8: Biotransformation of high farnesol using SHC variants. Reactions were carried out at A: 250 g / l EEH and SHC#65P2 and B: 300 g / l EEH and SHC#65, at different [EEH]:[cell] ratios, and T, pH, and SDS (SDS:cell ratio) conditions were defined as optimal conditions for each variant.

[0109] Figure 9: Amino acid sequence alignment of squalene-hopaene cyclases. This figure shows the amino acid sequence alignment of squalene-hopaene cyclases listed in Tables 3 and / or 16, prepared using CLUSTAL O (1.2.4). Amino acids at positions 169, 306, and 600 in the *Alicyclobacillus acidocaldarius* (AacSHC) sequence are highlighted in white against a black background. In the aligned amino acid sequences, W at position 169 is almost strictly conserved, as is G at position 600: 15 out of 16 sequences show only one conserved substitution. A at position 306 shows lower conservation (9 out of 16 sequences).

[0110] Figure 10 Cyclization reaction of bis(hoprofarniol) with SHC variants. The reaction occurs at 4 g / L E,E-bis(hoprofarniol) and OD... 650nm The study was conducted in 10 cells, using conditions defined as optimal T, pH, and SDS concentrations for SHC#65 and SHC#65P2, respectively.

[0111] Figure 11 The cyclization reaction of high-farnesol using wild-type and variant SHC enzymes. The reaction was carried out at 30°C in deionized water and 2.36 g / L high-farnesol and OD. 650nm It is carried out in 6 cells.

[0112] Figure 12 Bioconversion of homofarnesol using SHC#65P2. Reactions were performed with 400 g / l EEH and cells that had already produced SHC#65P2, using [cell]:[EEH] ratios of 0.6, 0.5, 0.4, and 0.3. Reactions were performed at 35°C, pH 5.8, and a constant [SDS]:[cell] ratio of 0.025.

[0113] Figure 13 Bioconversion of homofarnesol using SHC#65P2. Reactions were performed at 35°C, pH 5.8, and a constant [SDS]:[cells] ratio of 0.030 using either 450 g / l EEH and 180 g / l cells (a [cells]:[EEH] ratio of 0.4) or 250 g / l EEH and 250 g / l cells (a [cells]:[EEH] ratio of 1.0).

[0114] Sequence Summary

[0115] SEQ ID NO:1 is the amino acid sequence of wild-type Acidic Cyclocarya spp. (Aac) SHC.

[0116] SEQ ID NO:2 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A557T, and R613S, and may be referred to herein as SHC / HAC enzyme variant #65 or SHC#65 variant.

[0117] SEQ ID NO:3 can be called 215G2 SHC (or 215G2 SHC variant) and has M132R, A224V and I432T mutations corresponding to the wild-type AacSHC amino acid sequence.

[0118] SEQ ID NO:4 corresponds to SEQ ID NO:1 with the W169G substitution (also known as Aac SHC P1).

[0119] SEQ ID NO:5 corresponds to SEQ ID NO:1 with W169G and G600M substitutions (also known as Aac SHC P2).

[0120] SEQ ID NO:6 corresponds to SEQ ID NO:1 with the substitutions W169G, A306V, and G600M (also known as Aac SHCP3).

[0121] SEQ ID NO:7 corresponds to SEQ ID NO:2 with the W169G substitution (also known as the SHC#65P1 variant).

[0122] SEQ ID NO:8 corresponds to SEQ ID NO:2 with W169G and G600M substitutions (also known as the SHC#65P2 variant).

[0123] SEQ ID NO:9 corresponds to SEQ ID NO:2 with W169G, A306V and G600M substitutions (also known as the SHC#65P3 variant).

[0124] SEQ ID NO:10 corresponds to SEQ ID NO:3 with the W169G substitution (also known as the 215G2 SHC P1 variant).

[0125] SEQ ID NO:11 corresponds to SEQ ID NO:3 with W169G and G600M substitutions (also known as the 215G2 SHC P2 variant).

[0126] SEQ ID NO:12 corresponds to SEQ ID NO:3 with W169G, A306V and G600M substitutions (also known as the 215G2SHC P3 variant).

[0127] Wild-type SHC enzymes SEQ ID NO:13 to SEQ ID NO:34 and SEQ ID NO:384 may be used in this application as SEQ ID NO:1 (see also Tables 3 and 16). SEQ ID NO:13 is identical to SEQ ID NO:384.

[0128] SEQ ID NO:35 is the nucleotide sequence encoding wild-type AacSHC.

[0129] SEQ ID NO:36 is the nucleotide sequence encoding the polypeptide (SHC#65 variant) of SEQ ID NO:2.

[0130] SEQ ID NO:37 is the nucleotide sequence encoding the 215G2 variant (SEQ ID NO:3).

[0131] SEQ ID NO:38 is the nucleotide sequence encoding AacSHC P1 represented by SEQ ID NO:4.

[0132] SEQ ID NO:39 is the nucleotide sequence encoding AacSHC P2 represented by SEQ ID NO:5.

[0133] SEQ ID NO:40 is the nucleotide sequence encoding AacSHC P3 represented by SEQ ID NO:6.

[0134] SEQ ID NO:41 is the nucleotide sequence encoding the SHC#65P1 variant represented by SEQ ID NO:7.

[0135] SEQ ID NO:42 is the nucleotide sequence encoding the SHC#65P2 variant represented by SEQ ID NO:8.

[0136] SEQ ID NO:43 is the nucleotide sequence encoding the SHC#65P3 variant represented by SEQ ID NO:9.

[0137] SEQ ID NO:44 is the nucleotide sequence encoding the 215G2 P1 variant represented by SEQ ID NO:10.

[0138] SEQ ID NO:45 is the nucleotide sequence encoding the 215G2 P2 variant represented by SEQ ID NO:11.

[0139] SEQ ID NO:46 is the nucleotide sequence encoding the 215G2 P3 variant represented by SEQ ID NO:12.

[0140] SEQ ID NO:47 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, Y81H, A557T, and R613S, and may be referred to herein as SHC / HAC enzyme variant #66.

[0141] SEQ ID NO:48 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, Y81H, H431L and A557T, and may be referred to herein as SHC / HAC enzyme variant #110B8.

[0142] SEQ ID NO:49 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, T90A and R613S, and may be referred to herein as SHC / HAC enzyme variant #90C7.

[0143] SEQ ID NO:50 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, A172T and M277K, and may be referred to herein as SHC / HAC enzyme variant #115A7.

[0144] SEQ ID NO:51 is the amino acid sequence of the SHC / HAC variant 215G2SHC (represented by SEQ ID NO:3) with the additional mutation L37Q.

[0145] SEQ ID NO:52 is the amino acid sequence of the SHC / HAC variant 215G2SHC (represented in SEQ ID NO:3) having the additional mutation V174I.

[0146] SEQ ID NO: 53 is the amino acid sequence of the SHC / HAC variant 215G2 SHC (represented in SEQ ID NO: 3) having the additional mutations V174I and F601Y.

[0147] SEQ ID NO: 54 is the amino acid sequence of the SHC / HAC variant 215G2 SHC (represented in SEQ ID NO: 3) having the additional mutations L37Q, V174I and F601Y.

[0148] SEQ ID NO:55-296, 307-310 are the wild-type amino acid sequences of SHC / HAC shown in Table 2 or 16.

[0149] SEQ ID NO:297-300 is the nucleotide sequence encoding SEQ ID NO:47-50.

[0150] SEQ ID NO: 301 is the amino acid sequence of a GmoSHC variant having V45L, Q54E, M184I, T326S, F624Y.

[0151] SEQ ID NO:302 is the amino acid sequence of TelSHC with mutations W172G, P311V, F425Y, and G609A.

[0152] SEQ ID NO: 303 is the amino acid sequence of ZmoSHC1 having Q221S, W222G, A368V, F486Y, G667A mutations.

[0153] SEQ ID NO:304 is the amino acid sequence of TelSHC with the W172G mutation (P1).

[0154] SEQ ID NO: 305 is the amino acid sequence of AacSHC having W169G, G600M, M132R, A224V, and I432T mutations.

[0155] SEQ ID NO: 306 is the amino acid sequence of AacSHC having W169G, G600M, M132R, and I432T mutations.

[0156] SEQ ID NOs: 307-310 (together with SEQ ID NOs: 55-296) are the wild-type amino acid sequences of the SHC / HACs identified in Tables 2 or 16.

[0157] SEQ ID NO: 311 is the amino acid sequence of ScoSHC1 with W196G mutation (P1)

[0158] SEQ ID NO: 312 is the amino acid sequence of ZmoSHC1 with the W222G mutation (P1).

[0159] SEQ ID NO: 313 is the amino acid sequence of ZmoSHC2 with a W177G mutation (P1).

[0160] SEQ ID NO: 314 is the motif DXDDTA found in SHC / HAC.

[0161] SEQ ID NOs: 315-360 are amino acid sequences of SHC variants, in particular SEQ ID NOs: 315-347, 348, 350-352 (except SEQ ID NO: 349) are SHC variants derived from AaC SHC (SEQ ID NO: 1).

[0162] SEQ ID NO: 315 corresponds to SEQ ID NO: 1 with the substitution W169G.

[0163] SEQ ID NO: 316 corresponds to SEQ ID NO: 1 with the substitution W169A.

[0164] SEQ ID NO: 317 corresponds to SEQ ID NO: 1 with the substitution W169V.

[0165] SEQ ID NO: 318 corresponds to SEQ ID NO: 1 with substitutions W169G and G600A.

[0166] SEQ ID NO: 319 corresponds to SEQ ID NO: 1 with substitutions W169G and G600V.

[0167] SEQ ID NO:320 corresponds to SEQ ID NO:1 with the substitutions W169G and G600L.

[0168] SEQ ID NO:321 corresponds to SEQ ID NO:1 with the substitutions W169G and G600I.

[0169] SEQ ID NO:322 corresponds to SEQ ID NO:1 with the substitutions W169G and G600M.

[0170] SEQ ID NO:323 corresponds to SEQ ID NO:1 with substitutions of W169A and G600A.

[0171] SEQ ID NO:324 corresponds to SEQ ID NO:1 with substitutions of W169A and G600V.

[0172] SEQ ID NO:325 corresponds to SEQ ID NO:1 with the substitutions W169A and G600L.

[0173] SEQ ID NO:326 corresponds to SEQ ID NO:1 with the substitutions W169A and G600I.

[0174] SEQ ID NO: 327 corresponds to SEQ ID NO: 1 with substitutions W169A and G600M.

[0175] SEQ ID NO:328 corresponds to SEQ ID NO:1 with the substitutions W169V and G600A.

[0176] SEQ ID NO: 329 corresponds to SEQ ID NO: 1 with substitutions W169V and G600V.

[0177] SEQ ID NO: 330 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600L.

[0178] SEQ ID NO: 331 corresponds to SEQ ID NO: 1 with substitutions W169V and G600I.

[0179] SEQ ID NO:332 corresponds to SEQ ID NO:1 with the substitutions W169V and G600M.

[0180] SEQ ID NO:333 corresponds to SEQ ID NO:1 with substitutions of W169G, G600A and A306V.

[0181] SEQ ID NO: 334 corresponds to SEQ ID NO: 1 with substitutions W169G, G600V and A306V.

[0182] SEQ ID NO:335 corresponds to SEQ ID NO:1 with substitutions of W169G, G600L and A306V.

[0183] SEQ ID NO:336 corresponds to SEQ ID NO:1 with substitutions of W169G, G600I and A306V.

[0184] SEQ ID NO: 337 corresponds to SEQ ID NO: 1 with substitutions W169G, G600M and A306V.

[0185] SEQ ID NO: 338 corresponds to SEQ ID NO: 1 with substitutions W169A, G600A and A306V.

[0186] SEQ ID NO: 339 corresponds to SEQ ID NO: 1 with substitutions W169A, G600V and A306V.

[0187] SEQ ID NO: 340 corresponds to SEQ ID NO: 1 with substitutions W169A, G600L and A306V.

[0188] SEQ ID NO: 341 corresponds to SEQ ID NO: 1 with the substitutions W169A, G600I and A306V.

[0189] SEQ ID NO: 342 corresponds to SEQ ID NO: 1 with substitutions W169A, G600M and A306V.

[0190] SEQ ID NO: 343 corresponds to SEQ ID NO: 1 with substitutions W169V, G600A and A306V.

[0191] SEQ ID NO: 344 corresponds to SEQ ID NO: 1 with substitutions W169V, G600V and A306V.

[0192] SEQ ID NO: 345 corresponds to SEQ ID NO: 1 with substitutions W169V, G600L and A306V.

[0193] SEQ ID NO: 346 corresponds to SEQ ID NO: 1 with substitutions W169V, G600I and A306V.

[0194] SEQ ID NO:347 corresponds to SEQ ID NO:1 with substitutions of W169V, G600M and A306V.

[0195] SEQ ID NO: 348 corresponds to SEQ ID NO: 1 with substitutions M132R, A224V and I432T.

[0196] SEQ ID NO: 349 is a nucleotide sequence encoding an SHC variant derived from AacSHC.

[0197] SEQ ID NO: 350 corresponds to SEQ ID NO: 1 with substitutions W169G and A306V.

[0198] SEQ ID NO: 351 corresponds to SEQ ID NO: 1 with substitutions W169A and A306V.

[0199] SEQ ID NO:352 corresponds to SEQ ID NO:1 with the substitutions W169V and A306V.

[0200] SEQ ID NO:353 corresponds to SEQ ID NO:13 with substitutions of W222G and G667M (also known as the ZmoSHC1P2 variant).

[0201] SEQ ID NO:354 corresponds to SEQ ID NO:13 with the substitutions W222G, A368V and G667M (also referred to as ZmoSHC1 P3 variant).

[0202] SEQ ID NO:355 corresponds to SEQ ID NO:15 with the substitutions W177G and G619M (also known as the ZmoSHC2P2 variant).

[0203] SEQ ID NO:356 corresponds to SEQ ID NO:15 with substitutions of W177G, A321V and G619M (also known as the ZmoSHC2 P3 variant).

[0204] SEQ ID NO:357 corresponds to SEQ ID NO:32 with the substitutions W196G and G629M (also referred to as ScoSHC P2 variant).

[0205] SEQ ID NO:358 corresponds to SEQ ID NO:32 with the substitutions W196G, A335V and G629M (also referred to as ScoSHC P3 variant).

[0206] SEQ ID NO:359 corresponds to SEQ ID NO:23 with substitutions of W172G and G609M (also known as the TelSHC P2 variant).

[0207] SEQ ID NO:360 corresponds to SEQ ID NO:23 with substitutions of W172G, A311V and G609M (also known as TelSHC P3 variant).

[0208] SEQ ID NO:361-383 are the amino acid sequences of the SHC variants derived from Aac SHC (SEQ ID NO:1) shown in Table 18.

[0209] SEQ ID NO:361 corresponds to SEQ ID NO:1 with the substitution T77A.

[0210] SEQ ID NO:362 corresponds to SEQ ID NO:1 with the substitution I92V.

[0211] SEQ ID NO:363 corresponds to SEQ ID NO:1 with the substitution F129L.

[0212] SEQ ID NO:364 corresponds to SEQ ID NO:1 with the M132R substitution.

[0213] SEQ ID NO:365 corresponds to SEQ ID NO:1 with the substitution A224V.

[0214] SEQ ID NO:366 corresponds to SEQ ID NO:1 with the substitution I432T.

[0215] SEQ ID NO:367 corresponds to SEQ ID NO:1 with the substitution Q579H.

[0216] SEQ ID NO:368 corresponds to SEQ ID NO:1 with the substitution F601Y.

[0217] SEQ ID NO:369 corresponds to SEQ ID NO:1 with the substitutions M132R and I432T.

[0218] SEQ ID NO:370 corresponds to SEQ ID NO:1 with the substitution F601Y.

[0219] SEQ ID NO:371 corresponds to SEQ ID NO:1 with substitutions of T77A, I92V and F129L.

[0220] SEQ ID NO:372 corresponds to SEQ ID NO:1 with the substitutions Q579H and F601Y.

[0221] SEQ ID NO: 373 corresponds to SEQ ID NO: 1 with the substitution of F129L.

[0222] SEQ ID NO:374 corresponds to SEQ ID NO:1 with the substitutions F129L and F601Y.

[0223] SEQ ID NO:375 corresponds to SEQ ID NO:1 with the substitutions F129L, M132R and I432T.

[0224] SEQ ID NO:376 corresponds to SEQ ID NO:1 with the substitutions M132R, I432T and F601Y.

[0225] SEQ ID NO: 377 corresponds to SEQ ID NO: 1 with substitutions F129L, M132R, I432T and F601Y.

[0226] SEQ ID NO: 378 corresponds to SEQ ID NO: 1 with the substitution F605W.

[0227] SEQ ID NO: 379 corresponds to SEQ ID NO: 1 with substitutions of M132R, A224V, I432T, A557T, and H431L, and may be referred to herein as SHC / HAC enzyme variant #49 or SHC #49 variant.

[0228] SEQ ID NO: 380 corresponds to SEQ ID NO: 1 with substitutions M132R, A224V, I432T, Y81H, A557T, and R613S, and may be referred to herein as SHC / HAC enzyme variant #66 or SHC #66 variant.

[0229] SEQ ID NO: 381 corresponds to SEQ ID NO: 1 with substitutions M132R, A224V, I432T, Y81H, H431L, and A557T, and may be referred to herein as SHC / HAC enzyme variant #110B8 or SHC #110B8 variant.

[0230] SEQ ID NO: 382 corresponds to SEQ ID NO: 1 with substitutions M132R, A224V, I432T, T90A, and R613S, and may be referred to herein as SHC / HAC enzyme variant #90C7 or SHC #90C7 variant.

[0231] SEQ ID NO:383 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, A172T and M277K, and may be referred to herein as SHC / HAC enzyme variant #115A7 or SHC#115A7 variant.

[0232] SEQ ID NO: 385 corresponds to SEQ ID NO: 3 with substitutions W169G and A306V.

[0233] SEQ ID NO: 386 corresponds to SEQ ID NO: 2 with substitutions W169G and A306V. Detailed Description of the Invention

[0235] SHC / HAC enzymes and their variants

[0236] As used herein, the term "SHC enzyme" refers to wild-type (WT) squalene-hopaene cyclase, which is naturally present in, for example, thermophilic bacteria such as Alicyclobacillus acidocaldarius (Aac). SHC that functions in the cyclization of homofarnesol to ambroxol may also be referred to as homofarnesol-ambroxol cyclization (HAC) enzyme. Thus, the term "SHC / HAC enzyme" may be used herein.

[0237] As used herein, the term "variant" should be understood as a polypeptide that differs from its source polypeptide due to one or more changes or alterations in its amino acid sequence. The polypeptide from which a variant is derived is also called the parent or reference polypeptide. Typically, variants are artificially constructed, preferably by genetic engineering techniques. Typically, the polypeptide from which a variant is derived is a wild-type protein or a wild-type protein domain. However, variants available in this disclosure may also be derived from homologs, orthologs, or paralogs of the parent polypeptide, or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. Changes in the amino acid sequence can be amino acid exchanges (substitutions), insertions, deletions, N-terminal truncation, or C-terminal truncation, or any combination of these changes, which may occur at one or more sites.

[0238] As used herein, the term "SHC / HAC enzyme variant" refers to an enzyme that is derived from a wild-type SHC enzyme (e.g., Aac SHC enzyme, represented by SEQ ID NO: 1) but has one or more amino acid changes compared to the wild-type SHC enzyme, and therefore does not naturally occur in prokaryotes. Table 2 provides a list of wild-type SHCs that can be used in the present application to generate SHC / HAC enzyme variants. Tables 3 and 16 provide a list of preferred wild-type SHCs that can be used in the present application to generate SHC / HAC enzyme variants. Table 3 lists the following SHC / AHC enzymes: AaC SHC represented by SEQ ID NO: 1, ZmoSHC1 represented by SEQ ID NO: 13 or 384, ZmoSCH2 represented by SEQ ID NO: 15, BjpSHC represented by SEQ ID NO: 16, Burkhoderia ambifaria SHC represented by SEQ ID NO: 17 or 18, Bacillus anthracis SHC represented by SEQ ID NO: 19, Frankia alni SHC represented by SEQ ID NO: 20, Rhodopseudomonas palustris SHC represented by SEQ ID NO: 21, GmoSCOSHC represented by SEQ ID NO: 22, Tel SHC represented by SEQ ID NO: 23, ApaSHC1 represented by SEQ ID NO: 24, BmeSHC represented by SEQ ID NO: 25, SalSHC represented by SEQ ID NO: 26, ApaSHCA represented by SEQ ID NO: 27, BamSHC1 represented by SEQ ID NO: 28, BamSHC2 represented by SEQ ID NO: 29, PcaSHC2 represented by SEQ ID NO: 30 (or Syntrophotalea carbinolica DSM 2380), RpaSHC1 represented by SEQ ID NO: 31, ScoSHC represented by SEQ ID NO: 32, SfuSHC represented by SEQ ID NO: 33, and TtuSHC represented by SEQ ID NO: 34. More preferred wild-type SHCs used to generate SHC / HAC enzyme variants in the present application are: AaC SHC represented by SEQ ID NO: 1, ZmoSHC1 represented by SEQ ID NO: 13, ZmoSCH2 represented by SEQ ID NO: 15, Tel SHC represented by SEQ ID NO: 23, and ScoSHC represented by SEQ ID NO: 32.

[0239] One or more amino acid alterations can, for example, alter (e.g., increase) the enzyme activity against a substrate (e.g., EEH). In the context of this application, SHC / HAC enzyme variants can be derived from SHC / HAC variants. Example SHC / HAC variants include SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0240] This document describes assays for determining and quantifying the activity of SHC / HAC enzymes and / or SHC / HAC enzyme variants, methods known in the art. For example, the activity of SHC / HAC enzymes and / or SHC / HAC enzyme variants can be determined by incubating a purified SHC / HAC enzyme or enzyme variant, or an extract from a host cell or an intact recombinant host organism that produces the SHC / HAC enzyme or enzyme variant, with a suitable substrate under appropriate conditions, and analyzing the reaction products (e.g., by gas chromatography (GC) or HPLC). Further details regarding the assays of SHC / HAC enzyme and / or SHC / HAC enzyme variant activity and the analysis of reaction products are provided in the examples. The assays involve producing an SHC / HAC enzyme variant in a recombinant host cell (e.g., *E. coli*).

[0241] As used herein, the term "activity" refers to the ability of an enzyme to react with a substrate to provide a desired product. Activity can be determined in so-called activity assays to monitor the formation of the desired product. The SHC / HAC enzyme derivatives of this disclosure are characterized by their ability to cyclize homofarethol (e.g., EEH) to (-)-ambroside and exhibit biological activities, such as HAC activity. The SHC / HAC enzyme derivatives of this disclosure are also characterized by their ability to cyclize dihomorethol (e.g., E,E-dihomorethol) to ambergris oxide.

[0242] In the context of this application, the activity or biological activity of an SHC / HAC variant is compared to the corresponding activity or biological activity of the wild-type, parent, reference SHC / HAC from which it is derived under the same conditions. Exemplary wild-type SHC / HAC enzymes are identified in Tables 2, 3, or 16. In one embodiment, the wild-type SHC / HAC enzyme is represented by SEQ ID NO: 1 or by SEQ ID NO: IDNO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,6 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 10 2, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 , 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210 、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、Any one of 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310. Exemplary parent or reference SHC / HAC enzymes used to generate the SHC / HAC variants of the present invention can be represented by any one of SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0243] As used herein, "biological activity" refers to any activity that a polypeptide can exhibit, including but not limited to: enzymatic activity; substrate specificity, selectivity (e.g., substrate selectivity and / or product selectivity), conversion rate (e.g., EEH:EZH conversion rate), yield, binding activity to another compound (e.g., binding to another polypeptide, particularly to a receptor, or to a nucleic acid); inhibitory activity (e.g., enzyme inhibitory activity); activation activity (e.g., enzyme activation activity); or toxic effect. Variants are not required to exhibit such activities to the same extent as the parent or wild-type or reference polypeptide. A variant is considered a variant in the context of this application if it exhibits the relevant activity to an extent that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or at least 200% of the activity of the parent polypeptide. Likewise, a variant is considered a variant in the context of this application (as the terms derivative and variant are used interchangeably throughout this disclosure) if it exhibits the relevant biological activity to a degree that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or at least 200% of the activity of the parent polypeptide.

[0244] In one embodiment, the SHC / HAC enzyme variant described herein exhibits a better yield (i.e., increased yield) compared to the yield obtained using a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, such as AacSHC / AHC enzyme or a known SHC / HAC enzyme variant or a parental SHC from which a variant is derived). The term "yield" refers to the number of grams (which can be calculated as a percentage of molar conversion) of recyclable product (i.e., (-)-ambroxol or ambergris oxide) per gram of feedstock. In this document, "increased" can refer to an increase in yield of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% using the reference SHC enzyme.

[0245] In one embodiment, "selectivity" refers to "product selectivity" and describes the ability to produce a specific compound (e.g., (-)-ambroxol or ambergris oxide) from a mixture of several substrate isomers by an enzymatic catalytic method in a significantly enriched / dominant form ("product selectivity"). In one embodiment, the high farnesol substrate isomer mixture is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and / or [(3E,7E) and (3E,7Z)]. In one embodiment, the mixture is [(3E,7E), (3Z,7E)], also referred to as [EE:EZ] or EEH:EZH. In one embodiment, the EEH:EZH ratio in the mixture may be about 50:50 to about 99:01, or about 60:40 to about 99:1, or about 70:30 to about 95:5, or about 80:20 to about 95:5.

[0246] In preferred embodiments, "selectivity" refers to "product selectivity" and describes the ability to produce a specific compound (e.g., (-)-Ambroxol, i.e., Compound (I)) in a significantly enriched / major form ("product selectivity") from an EEH:EZH mixture (in a ratio of 80:20 or 90:10) in an enzymatic process.

[0247] In this article, "significant enrichment / major form" can refer to the total product formed due to the cyclization reaction of the enzymatic variant of WT SHC, which may consist essentially of the following substances or be composed of the following substances:

[0248] - when any of the above homofarnesol substrate isomer mixtures are used (preferably EEH:EZH, more preferably in a ratio of 80:20 or 90:10), the compound of formula (I) ((-)-Ambroxol) described herein, or

[0249] - When using any of the above high farnesol substrate isomer mixtures (preferably EEH:EZH, more preferably 80:20 or 90:10 ratio), the compounds of formula (I) ((-)-ambroxan) and formula (III) described herein.

[0250] This means that the compounds of formula (II) and (IV) may not be detectable at the end of the process or within the first 3, 4, 5 or 6 or 12 or 18 hours after the start of the process. Detection of the compounds of formula (I), (II), (III) and (IV) may be carried out using techniques known to those skilled in the art, preferably using the techniques used in the Examples section. This also means that only the compound of formula (I) or only the compounds of formula (I) and (III) are produced without any downstream processing steps. This means that downstream processing (DSP) can be simplified because the degree of substrate conversion and the substrate conversion rate are increased. In other words, DSP is simplified because the amount of unreacted homofarnesol present at the end of the reaction can be reduced. It is known that unreacted homofarnesol dissolves at least a portion of the solid (-)-Ambroxol formed, and that the solid (-)-Ambroxol formed may also be soluble in other liquid by-products, and if these by-products are not present (e.g. Compound II and / or IV), the DSP step can be simplified.

[0251] As used herein, "downstream processing steps" refer to the separation of solid (-)-Ambroxol from the unreacted homofarnesol substrate, and the separation of (-)-Ambroxol from other by-products (e.g., Compounds II and / or III and / or IV). The DSP steps may include, but are not limited to, one or more of centrifugation, filtration, steam or organic solvent extraction or distillation or selective crystallization steps. As an example of simplifying DSP herein, a distillation step is more effective in separating (-)-Ambroxol from unreacted homofarnesol than separating (-)-Ambroxol from the by-products produced. Each of these downstream processing steps is defined later herein. Example downstream processing steps are provided in WO2022 / 023464, the contents of which are incorporated herein by reference.

[0252] In one embodiment, a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, such as the AacSHC / AHC enzyme represented by SEQ ID NO:1, or by SEQ ID NO:1) is used. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70 ,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,Any other wild-type SHC / HAC enzyme represented by SEQ ID NO: 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or any known variant of an SHC / HAC enzyme, or a parental SHC from which a variant is derived, such as those represented by SEQ ID NO: 281, 282, 283, 284, 285, 286, 287, 288, 289, 310. The SHC / HAC enzyme variants described herein exhibit increased product selectivity (i.e., increased selectivity for (-)-Ambroxol or Ambergris oxide) compared to the product selectivity obtained with any of the SHC / HACs represented by NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. The term "product selectivity" also refers to the grams of recoverable product (i.e., (-)-Ambroxol or Ambergris oxide) per gram of total product formed (which can be calculated as a molar conversion percentage). In this document, "increase" can refer to an increase in yield obtained using the reference SHC enzyme of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400%.

[0253] In one embodiment, "selectivity" refers to the ability to preferentially convert a specific substrate isomer (e.g., EEH isomer) to a specific product from a mixture of multiple / several substrate isomers in an enzymatic process described herein ("substrate selectivity"). More specifically, this means that the specific product (-)-Ambrox is enriched relative to the enzymatic conversion of a specific substrate (EEH) isomer from a mixture of several substrate isomers.

[0254] Therefore, the “substrate selectivity” of an enzyme or enzyme variant refers to the ability of an enzyme or enzyme variant to react with a specific substrate isomer rather than another when the enzyme is contacted with a mixture containing at least two different substrate isomers. For example, WT SHC enzymes (e.g., those specified by SEQ ID NO:1, or any SHC / HAC disclosed in Tables 2, 3, or 16, or those specified by SEQ ID NO:1) NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67, 68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,1 03, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 15 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310), or an enzyme variant of WT SHC (e.g., represented by SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383), is selective for EEH over other homofarnesol isomers, or is selective for BisEEH over other dihomofarnesol isomers, meaning that the WT SHC enzyme or the enzyme variant of WT SHC is more likely to convert EEH than other homofarnesol isomers, or is more likely to convert BisEEH than other dihomofarnesol isomers. In one embodiment, the mixture of homofarnesol substrate isomers comprising at least two different homofarnesol substrate isomers comprises an EEH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and / or [(3E,7E) and (3E,7Z)]. In one embodiment, the mixture is [(3E,7E), (3Z,7E)], also referred to as [EE:EZ] or EEH:EZH. In one embodiment, the ratio of EEH:EZH in the mixture can be about 55:45, 50:50 to about 99:01, or about 60:40 to about 99:1, or about 70:30 to about 95:5, or about 80:20 to about 95:5.

[0255] For example, the wt% of the total product formed by reacting an enzyme variant of a WT, reference, or parental SHC with a mixture containing at least two different highfarnesol substrate isomers (the mixture containing EEH) may be at least about 1 percentage point higher than the wt% of the total product formed by reacting the corresponding WT, parental, or reference SHC enzyme with the same mixture containing EEH. For example, the wt% of the total product formed by reacting the enzyme variant may be at least about 2 percentage points higher, or at least about 3 percentage points higher, or at least about 4 percentage points higher than the wt% of the total product formed by reacting the WT, reference, or parental SHC with the same mixture containing EEH. For example, the wt% of the total product formed by reacting the enzyme variant may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points higher than the wt% of the total product formed by reacting the WT, reference, or parental SHC with the same mixture containing EEH. For example, the wt% of the total product formed by reacting the enzyme variant with a mixture containing EEH may be about 1 to about 40, or about 2 to about 30, or about 3 to about 20, or about 4 to about 10 percentage points higher than the wt% of the total product formed by reacting the WT SHC or the reference or parental SHC with the same mixture containing EEH. When using a mixture containing at least two different high farnesol substrate isomers (the mixture containing EEH) as a substrate, the total product formed by reacting the enzyme variant of WT SHC or the reference or parental SHC may, for example, consist of, substantially, or compounds of formula (I) ((-)-ambroside) and formula (IV) as described herein. In all these embodiments, the mixture of highfarnesol substrate isomers comprising at least two different highfarnesol substrate isomers comprises the EEH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and / or [(3E,7E) and (3E,7Z)]. In one embodiment, the mixture is [(3E,7E), (3Z,7E)], also referred to as [EE:EZ] or EEH:EZH. In one embodiment, the EEH:EZH ratio in the mixture may range from about 55:45, 50:50 to about 99:01, or about 60:40 to about 99:1, or about 70:30 to about 95:5, or about 80:20 to about 95:5.

[0256] When bisEEH is used as a substrate, the total product formed by the reaction of an enzyme variant of WT or a reference or parental SHC may, for example, consist substantially of or be composed of compounds of formula (X) and / or formula (XII) as described herein.

[0257] For example, using WT SHC / HAC (e.g., represented by SEQ ID NO:1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 307, 308, 309 or 310) or a reference / variant SHC / HAC (e.g., represented by SEQ ID 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383) may, for example, have an wt% of (-)-Ambroxol formed by an enzyme variant comprising at least about 1 percentage point higher than the wt% of (-)-Ambroxol formed by the reaction of the WT or reference / variant SHC with a mixture comprising at least two different homofarnesol substrate isomers, the mixture comprising EEH. For example, the wt% of (-)-Ambroxol formed by reacting the enzyme variant of the WT or reference or parent SHC with the mixture comprising EEH may be at least about 2 percentage points, or at least about 3 percentage points, or at least about 4 percentage points higher than the wt% of (-)-Ambroxol formed by reacting the WT or reference or parent SHC with the mixture comprising EEH. For example, the wt% of (-)-Ambroxol formed by reacting the enzyme variant of the WT SHC with the mixture comprising EEH may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points higher than the wt% of (-)-Ambroxol formed by reacting the WT or the reference or parent SHC with the mixture comprising EEH. For example, the wt% of (-)-Ambroxol formed upon reaction of the WT or the enzyme variant of the parent or reference SHC with the mixture comprising EEH may be about 1 to about 40, or about 2 to about 30, or about 3 to about 20, or about 4 to about 10 percentage points higher than the wt% of (-)-Ambroxol formed upon reaction of the WT or the reference or parent SHC with the mixture comprising EEH. In all of these embodiments, the mixture of homofarnesol substrate isomers comprising at least two different homofarnesol substrate isomers comprises EEH isomers and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and / or [(3E,7E) and (3E,7Z)]. In one embodiment, the mixture is [(3E,7E), (3Z,7E)], also referred to as [EE:EZ] or EEH:EZH. In one embodiment, the ratio of EEH:EZH in the mixture can be about 55:45, 50:50 to about 99:01, or about 60:40 to about 99:1, or about 70:30 to about 95:5.Or approximately 80:20 to approximately 95:5.

[0258] WT SHC / HAC (e.g., represented by SEQ ID NO: 1 or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 10 9, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170 , 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 2 62, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,The selectivity of enzyme variants of the parent or reference SHC / HAC (e.g., those represented by SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) can also be compared by the EEH:EZH conversion rate (i.e., EEH conversion rate:EZH conversion rate) or bisEEH:bisEZH conversion rate (i.e., bisEEH conversion rate:bisEZH conversion rate) of the reaction using each enzyme with the corresponding WT. The selectivity of SHC / HAC or the corresponding reference / parental SHC / HAC enzymes can be compared. This can be determined by measuring the amount of EEH and EZH or bisEEH and bisEZH remaining in the reaction mixture at the end of the reaction. The reaction is usually completed after 18 or 20 hours. Optionally, WT SHC / HAC (e.g., by SEQ ID NO: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79) can be compared. , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 1 21, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 Those represented by SEQ ID NO(251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310) or references or variants of SHC / HAC (e.g., those represented by SEQ ID NO(251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310) or references or variants of SHC / HAC (e.g., those represented by SEQ ID NO(251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 2 The selectivity of enzyme variants (ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) can also be compared by comparing the proportions of products generated by conversion with EEH (compounds of formulas I and IV) and EZH (compounds of formulas II and III), or by conversion with bisEEH (compounds of formulas X and XII) and bisEZH (compounds of formulas XI and XIII), to the selectivity of the corresponding WT or reference or parental SHC / HAC.

[0259] WT or reference or parental SHC / HAC enzyme variants can, for example, provide an EEH:EZH conversion of at least about 2.0 in the preparation of (-)-ambroxol from a mixture of at least two different high-farnesol substrate isomers, said mixture comprising EEH and EZH. In one embodiment, the high-farnesol substrate isomer mixture comprises EEH and EZH isomers and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E) and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)] and / or [(3E,7E) and (3E,7Z)]. In one embodiment, the mixture comprises EEH and EZH. In one embodiment, the mixture is [(3E, 7E), (3Z, 7E)], also referred to as [EE:EZ] or EEH:EZH. In one embodiment, the ratio of EEH:EZH in this mixture may range from about 55:45, 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0260] For example, in a method for preparing (-)-ambroxol from a mixture as defined above, an enzyme variant of WT or the reference or parental SHC / HAC can provide an EEH:EZH conversion rate of at least about 2.5, or at least about 3.0, or at least about 3.5. In one embodiment, the mixture comprises EEH and EZH. For example, in a method for preparing (-)-ambroxol from a mixture as defined above, the WT SHC / HAC or the enzyme variant of the reference or parental SHC / HAC can provide an EEH:EZH conversion rate of up to about 5.0, or up to about 4.5, or up to about 4.0. In one embodiment, the mixture comprises EEH and EZH. For example, in a method for preparing (-)-ambroxol from a mixture as defined above, the WT or the enzyme variant of the reference or parental SHC / HAC can provide an EEH:EZH conversion rate in the range of about 2.0 to about 5.0, or about 2.5 to about 4.5, or about 3.0 to about 4.0. In one embodiment, the mixture comprises EEH and EZH. This can be contrasted with the conversion provided by AacSHC in a process for preparing (-)-Ambrox from a mixture comprising EEH and EZH, for example, which can be less than about 2.0.

[0261] Specifically, when a WT SHC enzyme or a variant of WT SHC exhibits higher selectivity for EEH than other isomers of high-farnesol compared to variants of WT AacSHC and / or WT AacSHC, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be selected from TelSHC1, ApaSHC1, ZmoSHC1, ZmoSHC2, BjaSHC (or BjpSHC), GmoSHC, BmeSHC, SalSHC, and ApaSHCA. For example, when a WT SHC enzyme or a variant of WT SHC exhibits higher selectivity for EEH than other isomers of high-farnesol compared to variants of WT AacSHC and / or WTAacSHC, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be selected from ZmoSHC1, BjaSHC (BjpSHC), GmoSHC, ApaSHC1, and BmeSHC.

[0262] In one embodiment, the enzyme is compared to a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, such as that represented by SEQ ID NO:1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 08, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 1 68, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,The SHC / HAC enzyme variants described herein exhibit increased EEH conversion (or increased BisEEH conversion) compared to the conversion rates of known SHC / HAC enzyme variants or parental SHCs from which such variants are derived (e.g., those represented by SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). The term "conversion rate" refers to the amount of substrate (i.e., EEH or BisEEH) converted per gram of biocatalyst and per unit time (which can be calculated as a molar percentage of conversion). In this document, "increase" can refer to an increase in conversion obtained using a reference SHC enzyme of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400%.

[0263] In one embodiment, the enzyme is compared to a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, such as that represented by SEQ ID NO:1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 08, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 1 68, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,The SHC / HAC enzyme variants described herein exhibit increased selectivity for their substrate EEH (or BisEEH) compared to those represented by SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 for the same substrate. The term "substrate specificity" also refers to the amount of a specific converted substrate isomer (i.e., EEH or BisEEH) of each gram of initially present substrate at a certain time point of the enzyme-catalyzed reaction or during an "interval" of the reaction. In particular, the selectivity can be observed during an "interval" of 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15 to 75%, 20 to 70%, 25 to 65%, 30 to 60%, or 40 to 50% conversion of the initial amount of substrate. In one embodiment, the "interval" can be 1 to 8 hours, 2 to 7 hours, or 3 to 6 hours. In one embodiment, the "interval" is 6 hours. In this document, "increase" can refer to an increase in substrate specificity obtained using a reference SHC enzyme of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or 400%.

[0264] In further embodiments, the variant is derived from a parent SHC enzyme relative to a reference SHC enzyme (e.g., wild-type AacSHC (SEQ ID NO: 1) or 215G2 AacSHC (SEQ ID NO: 3) or SHC#65 (SEQ ID NO: 2), such as any of the following wild-type SHC / HACs: those identified in Tables 2 or 3 or 16 or those identified by SEQ ID NO: 1. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67 ,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,10 2、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 9, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,SEQ IDs 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, or derived from the following SEQ IDs. Any variant of NO: 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the SHC / HAC enzyme variants described herein exhibit altered (e.g., increased) productivity. The term "productivity" refers to the amount of product (i.e., (-)-Ambroxol or Ambergris Oxide) that can be recovered in grams per liter of reaction volume per hour of bioconversion time (i.e., time after addition of substrate). The term "productivity" also refers to the amount of product (i.e., grams) that can be recovered in grams per liter of reaction volume per hour of bioconversion time (i.e., time after addition of substrate) per gram of biocatalyst used in the reaction. In this document, "increase" can refer to a productivity increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or 400% obtained using the reference SHC enzyme.

[0265] In one embodiment, the SHC / HAC enzyme variants disclosed herein exhibit:

[0266] - Increased substrate specificity for EEH (or BisEEH) when using homofarnesol or dihomofarnesol substrates,

[0267] - when homofarnesol or dihomofarnesol substrates are used, the product selectivity towards (-)-ambroxol (or ambergris oxide) is increased,

[0268] - When using substrates other than perfarnesol or bisperfarnesol (e.g., ethyl-perfarnesol, hydroxyfarnesol, etc. - see, for example, WO2021 / 110858 and WO2021 / 209482 respectively), the specificity for specific isomers of the substrate increases.

[0269] -Increased productivity, and / or

[0270] - During the first 3 to 6 hours of the reaction (or the first 4, 5, or 6 hours), the degree of conversion of EEH (or BisEEH) and the conversion rate of EEH (or BisEEH) increase.

[0271] The increase is relative to a reference SHC enzyme (e.g., wild-type SHC, such as that derived from SEQ ID). NO:1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 7 1, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9 1, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,Those represented by SEQ ID NO: 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or 215G2 AacSHC, or SHC#65, or parental SHC enzymes from which variants are derived, such as those represented by SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0272] In one embodiment, the SHC / HAC enzyme variants disclosed herein exhibit:

[0273] - increased substrate specificity for EEH (or for BisEEH) when using homofarnesol or dihomofarnesol substrates,

[0274] - when homofarnesol or dihomofarnesol substrates are used, the product selectivity towards (-)-ambroxol (or ambergris oxide) is increased,

[0275] - When using substrates other than perfarnesol or bisperfarnesol (e.g., ethyl-perfarnesol, hydroxyfarnesol, etc. - see, for example, WO2021 / 110858 and WO2021 / 209482 respectively), the specificity for specific isomers of the substrate increases.

[0276] -Increased productivity, and

[0277] - an increase in the degree of conversion of EEH (or BisEEH) and an increase in the conversion rate within the first 3 to 6 hours (or within the first 4, 5 or 6 hours) of the reaction,

[0278] The increase is compared to a reference SHC enzyme (eg, wild-type AacSHC or 215G2 AacSHC or SHC#65 or the parent SHC enzyme from which the variant is derived).

[0279] In further embodiments, the SHC is expressed as a nucleotide sequence of the present invention, as compared to a reference SHC enzyme (e.g., wild-type AacSHC (SEQ ID NO: 1) or SHC#65 (SEQ ID NO: 2), 215G2 AacSHC (SEQ ID NO: 3), ZmoSHC1 represented by SEQ ID NO: 13, ZmoSCH2 represented by SEQ ID NO: 15, BjpSHC represented by SEQ ID NO: 16, Burkhoderia ambifaria SHC represented by SEQ ID NO: 17 or 18, Bacillus anthracis SHC represented by SEQ ID NO: 19, Frankia alni SHC represented by SEQ ID NO: 20, Rhodopseudomonas palustris SHC represented by SEQ ID NO: 21, GmoSCOSHC represented by SEQ ID NO: 22, TelSHC represented by SEQ ID NO: 23, or a nucleotide sequence of SEQ ID NO: 24). The SHC / HAC enzyme variants described herein show altered (i.e., increased) production of the SHC / HAC enzymes ApaSHC1 represented by SEQ ID NO: 24, BmeSHC represented by SEQ ID NO: 25, SalSHC represented by SEQ ID NO: 26, ApaSHCA represented by SEQ ID NO: 27, BamSHC1 represented by SEQ ID NO: 28, BamSHC2 represented by SEQ ID NO: 29, PcaSHC2 represented by SEQ ID NO: 30 (or Syntrophotalea carbinolica DSM 2380), RpaSHC1 represented by SEQ ID NO: 31, ScoSHC represented by SEQ ID NO: 32, SfuSHC represented by SEQ ID NO: 33, TtuSHC represented by SEQ ID NO: 34).

[0280] In a preferred embodiment, the SHC / HAC enzyme variants described herein exhibit altered (i.e., increased) yields compared to the yields of reference AacSHC (SEQ ID NO:1), or SHC#65 (SEQ ID NO:2), or 215G2 AacSHC (SEQ ID NO:3), or ZmoSHC1 represented by SEQ ID NO:13, or ZmoSCH2 represented by SEQ ID NO:15, or Tel SHC represented by SEQ ID NO:23, or ScoSHC represented by SEQ ID NO:32.

[0281] Specifically, the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant can be derived) can be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis (Zmo) SHC / HAC enzyme, a Bradyrhizobium japonicum (Bjp / Bja) SHC / HAC enzyme, an Acetobacter pasteurianus (Apa) SHC / HAC enzyme, a Bacillus megaterium (Bme) SHC / HAC enzyme, or a Gluconobacter morbifer (Gmo) SHC / HAC enzyme. Specifically, the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant can be derived) can be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme.

[0282] The term "target yield factor" refers to the ratio of the obtained product concentration to the concentration of the SHC / HAC variant enzyme in the reaction medium (e.g., a purified SHC / HAC enzyme variant or an extract from a recombinant host cell that produces the SHC / HAC enzyme variant). In various embodiments, the SHC / HAC enzyme variant disclosed herein is used relative to a reference SHC protein (e.g., SEQ ID NO). NO:1,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,6 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 1 57, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310 or 2, 3, 47, 48, 49, 50, 51 The activities of enzymes (e.g., 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) show altered (e.g., increased) enzyme activities by a fold increase (e.g., altered / increased high farnesol ambroxol cyclase (HAC) activity). This increase in activity can be at least by folds of: 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and / or 100.

[0283] As used herein, the term "amino acid change" refers to the insertion of one or more amino acids between two amino acids, the deletion of one or more amino acids, or the replacement (which may be conservative or non-conservative) of one or more amino acids with one or more amino acids that differ in amino acid sequence relative to a reference amino acid sequence. A substitution is the replacement of the amino acids of the reference sequence with the same number of amino acids in the variant sequence. The reference amino acid sequence may be, for example, a wild-type (WT) amino acid sequence (e.g., SEQ ID NO: 1 or SEQ ID NO: 2). NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,6 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10 0, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203 3, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), or may be, for example, an SHC / HAC enzyme variant sequence (e.g., Aac variant - SEQ ID). NO: 2 or 3 or 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383). The term "displacement" is synonymous with "substitution".

[0284] Amino acid changes can be readily identified by comparing the amino acid sequence of the SHC / HAC enzyme variant to that of a reference amino acid sequence.

[0285] For example, conserved amino acid substitutions can be performed based on the similarity of the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the amino acid residues involved. The above 20 naturally occurring amino acids can be divided into the following six standard amino acid groups:

[0286] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile;

[0287] (2) Neutral hydrophilicity: Cys, Ser, Thr; Asn, Gln;

[0288] (3) Acidity: Asp, Glu;

[0289] (4) Alkaline: His, Lys, Arg;

[0290] (5) Residues that affect chain orientation: Gly, Pro; and

[0291] (6) Aromatics: Trp, Tyr, Phe.

[0292] Therefore, the term "conservative substitution" as used herein refers to the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups mentioned above. For example, replacing Asp with Glu retains a negative charge in the polypeptide thus modified. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. Some preferred conservative substitutions within the six groups mentioned above are substitutions within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (iii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Based on the known genetic code and recombinant and synthetic DNA technology, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0293] In a preferred embodiment (as shown in Example 11), by referring to the wild-type AacSHC of SEQ ID NO:1, the amino acid sequence of wild-type SHC, particularly a group of conserved amino acids of the wild-type AacSHC of SEQ ID NO:1, includes L22, Q26, G30, W32, A44, L48, Q72, G76, W78, Y95, L98, G102, A113, I117, G121, G122, F129, T130, L134, A135, G138, W142, P146, W169. A170, R171, F217, D222, R237, I261, P263, P281, S309, P310, W312, D313, T314, A 320, W339, Q344, G349, D350, W351, G361, G362, A364, F365, N369, Y372, P373, D37 4. D376, D377, W406, Q411, G415, A419, P433, D436, D442, P443, D447, V448, Q479 , G483, W485, G487, R488, W489, G490, N492, Y495, G496, T497, L504, W522, Q527, G 531, G532, W533, G534, E535, S539, Y540, G547, T552, T556, W558, A559, A565, L581, Q585, G589, W591, G600, F601, P602, F605, Y609, Y612, F616, P617, A620, and R623.

[0294] In a preferred embodiment (as shown in Example 14), a set of conserved amino acids in the amino acid sequences of five wild-type SHCs is proposed, specifically wild-type AacSHC of SEQ ID NO: 1, wild-type ZmoSHC1 of SEQ ID NO: 13, wild-type ZmoSHC2 of SEQ ID NO: 15, wild-type TelSHC of SEQ ID NO: 23, and wild-type ScoSHC of SEQ ID NO: 32.

[0295] As used herein, “non-conservative substitution” or “non-conservative amino acid exchange” is defined as the exchange of one amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above. Typically, the SHC / HAC enzyme variants described herein are prepared using non-conservative substitutions that alter the biological function (e.g., HAC activity) of the disclosed SHC / HAC enzyme variants. For ease of reference, the single-letter amino acid symbols recommended by the IUPAC-IUB Biochemistry Nomenclature Committee are shown below. Three-letter codes are also provided for reference.

[0296] Table 1: Amino acid nomenclature

[0297] Single-letter codes Three-letter code Amino acid name A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu glutamate F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

[0298] Amino acid changes, such as amino acid substitutions, can be introduced using known recombinant gene technology protocols, including PCR, gene cloning, cDNA site-directed mutagenesis, host cell transfection, and in vitro transcription. These techniques can be used to introduce such changes into the reference sequence, thereby generating SHC / HAC enzyme variants. These enzyme variants can then be screened to test for SHC / HAC functional activity.

[0299] Suitable sources of SHC / HAC enzymes are listed in Tables 2, 3 or 16. In one embodiment, the suitable source includes, for example, *Bacillus cyclophosphamide* (Aac), *Zymomonas mobilis* (Zmo), *Bradyrhizobium japonicum* (Bjp), *Gluconobacter morbifer* (Gmo), *Burkholderia ambifaria*, *Bacillus anthracis*, *Methylococcus capsulatus*, *Frankia alni*, *Acetobacter pasteurianus* (Apa), *Thermosynechococcuselongatus* (Tel), *Streptomyces coelicolor* (Sco), *Rhodopseudomonas palustris* (Rpa), *Teredinibacter turnerae* (Ttu), and *Pelobacter*. *Syntrophotalea carbinolica* DSM 2380, *Bacillus megaterium* (Bme), *Streptomyces albolongus* (Sal), and *Tetrahymena pyriformis* (see, for example, WO 2010 / 139719, US2012 / 01345477, WO 2012 / 066059, the contents of which are incorporated herein by reference).

[0300] Specifically, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme variant can be derived from it) can be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis SHC / HAC (ZmoSHC1) enzyme, a Bradyrhizobium japonicum (Bjp or Bja) SHC / HAC enzyme, a Gluconobacter morbifer (Gmo) SHC / HAC enzyme, an Acetobacter pasteurianus SHC / HAC (ApaSHC1) enzyme, or a Bacillus megaterium (Bme) SHC / HAC enzyme. Specifically, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme variant can be derived from it) can be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme. The enzyme is represented by SEQ ID NO: 1.

[0301] For ease of reference, the name "AacSHC" may be used to refer to the Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, "ZmoSHC" may be used to refer to the Zymomonas mobilis (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" may be used to refer to the Bradyrhizobium japonicum (Bjp) SHC / HAC enzyme, "ApaSHC" may be used to refer to the Acetobacter pasteurianus (Apa) SHC / HAC enzyme, "BmeSHC" may be used to refer to the Bacillus megaterium (Bme) SHC / HAC enzyme, "SalSHC" may be used to refer to the Streptomyces albolongus (Sal) SHC / HAC enzyme, and "GmoSHC" may be used to refer to the Gluconobacter morbifer (Gmo) SHC / HAC enzyme.

[0302] The AacSHC, ZmoSHC, and BjpSHC enzyme sequences are disclosed in BASF WO 2010 / 139719, US 2012 / 01345477A1, Seitz et al. (as mentioned above), and Seitz (2012 doctoral dissertation, as mentioned above). Two different sequences of ZmoSHC, designated ZmoSHC1 and ZmoSHC2, are disclosed. The Gmo SHC / HAC enzyme sequence is disclosed in WO 2018 / 157021. The SalSHC enzyme is disclosed in Liu et al. (2020): A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene, Frontiers in Bioengineering and Biotechnology, vol 8, article 426, https: / / doi.org / 10.3389 / fbioe.2020.00426).

[0303] Table 2. Sources and accession numbers of wild-type (WT) SHC enzymes.

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318] Table 3. Sources and accession numbers of preferred wild-type (WT) SHC enzymes.

[0319]

[0320]

[0321]

[0322] The sequences of wild-type AacSHC, wild-type ZmoSHC1, wild-type ZmoSHC2, wild-type BjpSHC, wild-type GmoSHC, wild-type TelSHC, and wild-type ApaSHC1, wild-type BmeSHC, wild-type SalSHC, and wild-type ApaSHCA are also disclosed in Table 3.

[0323] The most preferred wild-type SHC is: AacSHC (SEQ ID NO: 1), or ZmoSHC1 represented by SEQ ID NO: 13, or ZmoSCH2 represented by SEQ ID NO: 15, or Tel SHC represented by SEQ ID NO: 23, or ScoSHC represented by SEQ ID NO: 32.

[0324] An alignment of WT SHC sequences prepared by Hoshino and Sato (2002, supra) showed that multiple motifs were detected, consisting of the core sequence Gln-XXX-Gly-X-Trp, which occurs six times in both Z. mobilis and Alicyclobacillus acidocaldarius SHC sequences (see Reipen et al. 1995, Microbiology 141, 155-161). Figure 3 (X can be any amino acid). Hoshino and Sato (2002, supra) reported that aromatic amino acids are unusually abundant in SHC and noted two characteristic motifs in SHC: a QW motif, represented by a specific amino acid sequence [(K / R)(G / A)X2-3(F / Y / W)(L / IV)3X3QX2-5GXW], and a DXDDTA motif (SEQ ID NO: 314). Wendt et al. (1997, Science 277, 1811-1815 and 1999, J Mol Biol 286, 175-187) reported X-ray structural analysis of Alicyclobacillus acidocaldarius SHC (X can be any amino acid). The DXDDTA motif appears to be associated with the SHC active site.

[0325] The reference AacSHC protein used in this article refers to the wild-type AacSHC protein disclosed in SEQ ID NO:1. AacSHC possesses hofarnesol ambroxol cyclase (HAC) activity and can be used to produce ambroxol derivatives via the biocatalytic reaction of SHC with hofarnesol substrates. The primary reaction of AacSHC is the cyclization of linear or nonlinear substrates (e.g., hofarnesol) to produce ambroxol.

[0326] Another reference AacSHC protein is the SHC / AHC enzyme 215G2 SHC or 215G2SHC disclosed as SEQ ID NO: 3. SEQ ID NO: 3 corresponds to SEQ ID NO: 1 with mutations M132R, A224V, and I432T.

[0327] Another reference AacSHC protein is the SHC / AHC enzyme variant #65 disclosed in SEQ ID NO:2. SEQ ID NO:2 corresponds to SEQ ID NO:1 with substitutions of M132R, A224V, I432T, A557T, and R613S, and may be referred to herein as SHC / HAC enzyme variant #65 or SHC#65 variant.

[0328] The term "functional homologue" may be replaced by "functional equivalent" or "homologue".

[0329] The functional homologs of wild-type SHC / HAC enzymes or SHC / HAC enzyme variants described herein are also suitable for cyclization reactions, such as for the production of (-)-ambroside, for example, in a recombinant host. Therefore, a recombinant host may include one or more heteronucleotides encoding the functional homologs of the aforementioned polypeptides and / or heteronucleotides encoding the SHC / HAC-derived enzymes described herein.

[0330] A functional homolog is a polypeptide that shares sequence identity with a reference polypeptide and optionally has sequence similarity, and performs one or more biochemical or physiological functions of the reference polypeptide. Both the functional homolog and the reference polypeptide can be naturally occurring polypeptides, and the sequence identity and optional sequence similarity may be due to convergent or divergent evolutionary events. Therefore, functional homologs are sometimes referred to in the literature as homologs, orthologs, or paralogs. Variants of naturally occurring functional homologs, such as polypeptides encoded by mutants of wild-type coding sequences, can themselves be functional homologs. Functional homologs can also be constructed by site-directed mutagenesis of the polypeptide coding sequence or by combining domains of coding sequences from different naturally occurring polypeptides (“domain exchange”). Techniques for modifying genes encoding the functional homologs described herein are known and include directed evolution techniques, site-directed mutagenesis techniques, and random mutagenesis techniques, and can be used to increase the specific activity of the polypeptide, alter substrate specificity, change expression levels, alter subcellular locations, or modify polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids that encode functional homologous polypeptides.

[0331] Functional homologs can be identified by analyzing nucleotide and peptide sequence alignments. For example, searching nucleotide or peptide sequence databases can identify homologs of nucleic acid sequences encoding SHC-derived peptides and their analogues.

[0332] Hybridization can also be used to determine functional homologs and / or as a measure of homology between two nucleic acid sequences. Nucleic acid sequences encoding any protein or portion thereof disclosed herein can be used as hybridization probes according to standard hybridization techniques. Hybridization of the probe with DNA or RNA from a detection source (e.g., mammalian cells) indicates the presence of the relevant DNA or RNA in the detection source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as equivalent to hybridization at 30°C in 2x sodium chloride / sodium citrate (SSC), followed by washing at 50°C in 1x SSC, 0.1% SDS. Highly stringent conditions are defined as equivalent to hybridization at 45°C in 6x sodium chloride / sodium citrate (SSC), followed by washing at 65°C in 0.2x SSC, 0.1% SDS. Sequence analysis to identify functional homologs may also involve using relevant amino acid sequences as reference sequences to perform BLAST, bidirectional BLAST, or PSI-BLAST analyses on a non-redundant database. In some cases, amino acid sequences are inferred from nucleotide sequences. Peptides with sequence identity greater than 40% in the database are candidates for further evaluation of their suitability for SHC / HAC biotransformation. Amino acid sequence similarity allows for conserved amino acid substitutions, such as substituting one hydrophobic residue for another or one polar residue for another. If desired, such candidates can be manually tested to narrow down the number of candidates to be further evaluated. Manual testing can be performed by selecting candidates that may possess, for example, conserved functional domains.

[0333] Typically, peptides exhibiting at least approximately 30% amino acid sequence identity can be used to identify conserved regions. The conserved regions of the relevant peptides exhibit at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, and 69% amino acid sequence identity. In some embodiments, the conserved region exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Sequence identity can be determined as described above and below. Typically, in this case, the region may contain 5 to 50 amino acids, 10 to 150 amino acids, 10 to 80 amino acids, or 10 to 100 amino acids.

[0334] The SHC / HAC enzyme or enzyme variant described herein and used in the methods described herein may, for example, be based on SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 ,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 16 4, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 2 22, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or an amino acid sequence of a variant, homologue, mutant, derivative, or fragment thereof.

[0335] In addition, the reference SHC enzyme produced can be based on the amino acid sequence produced by Escherichia coli (E. coli).

[0336] "Percent (%) identity" with respect to the nucleotide sequence of a gene is defined as the percentage of nucleotides in the candidate DNA sequence that are identical to the nucleotides in the DNA sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignments for determining percent nucleotide sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. The terms "polypeptide" and "protein" are used interchangeably herein to refer to a peptide chain linked by any amino acids, regardless of their length or post-translational modifications.

[0337] The term "derivative" as used herein includes, but is not limited to, variants. The terms "derivative" and "variant" are used interchangeably herein.

[0338] In preferred embodiments, the enzyme variants useful in the present disclosure exhibit a total of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200) changes (alterations) in the amino acid sequence (i.e., substitutions, insertions, deletions, N-terminal truncations and / or C-terminal truncations). The amino acid exchanges can be conservative and / or non-conservative. In preferred embodiments, variants useful in the present disclosure differ from the protein or domain from which they are derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid substitutions, preferably conservative amino acid changes. Variants may additionally or alternatively comprise amino acid deletions, which may be N-terminal truncations, C-terminal truncations or internal deletions or any combination thereof. In the context of the present application, such variants comprising N-terminal truncations, C-terminal truncations and / or internal deletions are referred to as "deletion variants" or "fragments". The terms "deletion variant" and "fragment" are used interchangeably herein. Deletion variants may be naturally occurring (e.g., splice variants) or artificially constructed, preferably by genetic engineering means. Typically, the protein or protein domain from which a deletion variant is derived is the wild-type protein. The present invention also can comprise the variant of the present invention or the variant of the present invention, wherein said variant comprises at least one amino acid whose N-terminal end and / or C-terminal end and / or internal end is missing or has 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 amino acids.

[0339] In certain embodiments, the SHC / HAC enzyme variants described herein comprise only substitutions and do not comprise any deletions or insertions.

[0340] As used herein, a "variant" may alternatively or additionally be characterized by having a degree of sequence identity or similarity to a parent polypeptide from which it is derived. A variant of the WT / reference SHC / HAC or SHC / HAC derivative of the present disclosure may have a sequence identity of at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 200%, %, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

[0341] The statement "at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%" is used. "77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity" is used throughout the specification with respect to polypeptide and polynucleotide sequence comparisons. Polynucleotides or proteins belonging to any family of enzymes disclosed herein can be identified based on their similarity to related genes or proteins, respectively. For example, identification can be based on sequence identity. In certain preferred embodiments, the disclosure features an isolated nucleic acid molecule that is identical to (a) a polypeptide encoding a wild-type SHC / HAC enzyme (e.g., SEQ ID NO: 1, SEQ ID NO: 2). NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 15 2, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 21 8, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 294, 295, 296, 307, 308, 309, 310, or any other SHC / HAC disclosed in Tables 2, 3, or 16 herein, (b) a nucleic acid molecule comprising a polypeptide of ID NO:35 and (c) a nucleic acid molecule comprising at least 30 (e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 850, 900, 950, 1000, or 1010) nucleotides of the nucleotide sequence encoding the SHC / HAC enzyme disclosed in Tables 2, 3, or 16, having at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 5 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0342] Preferably, the polypeptide and the reference polypeptide exhibit the aforementioned sequence identity or similarity over a continuous segment of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids. Preferably, the polynucleotide and the reference polynucleotide exhibit the aforementioned sequence identity over a continuous segment of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300, or more nucleotides. When comparing two sequences and no reference sequence is specified for calculating the percentage of sequence identity, the longer of the two sequences to be compared should be used for calculating sequence identity unless otherwise specified. If a reference sequence is specified, the sequence identity is calculated based on the reference sequence (e.g., SEQ ID). NO:1,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 41, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 18 7, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 27 Determine the full-length sequence identity of 1, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310 and any SHC / HAC enzyme disclosed in Tables 2, 3, or 16.

[0343] For example, a peptide sequence consisting of 130 amino acids can exhibit a maximum sequence identity percentage of 20.6% (130 / 631 x 100) compared to the full-length amino acid sequence of wild-type AacSHC with 631 amino acid residues, while a sequence of 300 amino acids can exhibit a maximum sequence identity percentage of 47.5% (300 / 631 x 100).

[0344] The identity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined by sequence alignment. Such alignment can be performed using a variety of algorithms known in the art, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), which can be obtained from, for example: https: / / www.ebi.ac.uk / Tools / msa / clustalo / , or the GAP program (mathematical algorithm of the University of Iowa), or the mathematical algorithm of Myers and Miller (1989-Cabios 4:11-17), or Clone Manager 9. The preferred parameters used are the default parameters as they are set at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0345] The rank of sequence identity (sequence match) can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed using the BLASTN program with score = 100 and word length = 12 to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins.

[0346] BLAST protein searches were performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the SHC peptide. For vacancy alignments used for comparative purposes, vacancy BLAST was employed, as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. The default parameters for both the BLAST and vacancy BLAST programs were used. Sequence matching analysis was supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are mentioned in this application, unless otherwise explicitly stated, these percentages are calculated relative to the full length of the longer sequence.

[0347] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0348] In one implementation, "similarity" refers to the degree of sequence association between amino acid sequences, which, depending on the case, is determined by matching strings of such sequences. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988)).

[0349] Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison Matrix: BLOSSUM 62 of Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. Programs using these parameters are publicly available in programs such as "Ogap" (from Genetics Computer Group, Madison, Wisconsin). The above parameters are the default parameters for amino acid comparisons (and no penalty for terminal gaps).

[0350] Alternatively, when determining amino acid similarity, those skilled in the art may also consider so-called “conservative” amino acid substitutions, which are readily apparent to them. The “similarity” between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues with similar side chains. The table below provides examples of categories of amino acid residues for conserved substitutions.

[0351] Table 4: Classes of amino acid residues

[0352] acidic residues Asp(D) and Glu(E) basic residues Lys(K), Arg(R), and His(H) Hydrophilic uncharged residues Ser(S), Thr(T), Asn(N) and Gln(Q) Aliphatic uncharged residues Gly(G), Ala(A), Val(V), Leu(L), and Ile(I) Nonpolar uncharged residues Cys(C), Met(M), and Pro(P) Aromatic residues Phe(F), Tyr(Y), and Trp(W)

[0353] Table 5: Substitution categories for conservative amino acid residues

[0354] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0355] Table 6: Classification of physical and functional substitutions of amino acid residues

[0356]

[0357]

[0358] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxy side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are those in which at least one residue in the disclosed sequence has been removed and a different residue has been inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each naturally occurring amino acid are as follows: Ala is replaced by Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser or Ala; Gln by Asn; Glu by Asp; Gly by Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln, or Glu; Met by Leu or Ile; Phe by Met, Leu, or Tyr; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; and Val by Ile or Leu.

[0359] Specific SHC / HAC enzymes and enzyme variants useful in the methods described herein are described further below.

[0360] SHC / HAC variants with novel mutations

[0361] Surprisingly, it was found that variants derived from the Aac SHC / HAC enzyme (SEQ ID NO: 1) or from variants of said Aac SHC / HAC enzyme (SEQ ID NO: 2 or SEQ ID NO: 3) or from other wild-type SHCs identified herein (e.g., those identified in Tables 2, 3 or 16, in particular variants such as those identified by SEQ ID NO: 1). NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67, 68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,1 03, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、47, 48, 49, 50, 51, 52, 53, 54) and those disclosed in Table 14 of WO2016 / 170099 and / or Table 6 of WO2021 / 110848 (provided below, the SHC / HAC enzyme variants are those represented by SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54) or derived from other variants (e.g., those represented by SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54) and those disclosed in Table 14 of WO2016 / 170099 and / or Table 6 of WO2021 / 110848 (provided below, the SHC / HAC enzyme variants are those represented by SEQ ID NO: 47, 48, 49, 50, 51, 52, 53, 54) or derived from other variants (e.g., those represented by SEQ ID NO: NO:361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383) SHC / HAC enzyme variants exhibiting the modified / optimized activity / biological activity previously defined herein and further demonstrated in the Examples section.

[0362] SEQ ID NO:3 corresponds to SEQ ID NO:1 with amino acid alterations (substitutions) M132R, A224V and I432T.

[0363] SEQ ID NO:2 corresponds to SEQ ID NO:1 with amino acid alterations (substitutions) M132R, A224V, I432T, A557T, and R613S.

[0364] In one embodiment, the novel SHC / HAC enzyme variants derived from SEQ ID NO:2 or 3, compared to SEQ ID NO:1, still contain amino acid alterations (substitutions). These amino acid alterations are as follows:

[0365] - Variants derived from SEQ ID NO:3 may include the following amino acid alterations (substitutions): M132R, A224, and I432T compared to SEQ ID NO:1.

[0366] - Variants derived from SEQ ID NO:2 may include the following amino acid alterations (substitutions): M132R, A224V, I432T, A557T, and R613S compared to SEQ ID NO:1.

[0367] - Variants derived from SEQ ID NO:2 may include the following amino acid alterations (substitutions): M132R and I432T compared to SEQ ID NO:1.

[0368] - A variant derived from SEQ ID NO: 2 may comprise the following amino acid changes (substitutions): M132R and I432T, A557T and R613S compared to SEQ ID NO: 1.

[0369] Variants derived from any of the sequences listed in Table 14 of WO2016 / 170099, including but not limited to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 171, 23, 25, 27, 29, 31, 33, 35, 37 or 39 (corresponding to SEQ ID NO: 361-368, 378, 3, 369-377 in the present application, respectively). (See Table 7 below and the sequences disclosed at the end of the Examples section).

[0370] Variations of any of the sequences listed in Table 6 of WO2021 / 110848, including but not limited to SEQ ID NO: 5, 17, 18, 2 or 4 (see Table 8 below and the sequences disclosed at the end of the Examples section).

[0371] Table 7: SEQ ID NO of amino acids and nucleotides of AacSHC derivatives corresponding to Table 14 of WO 2016 / 170099.

[0372]

[0373] Table 8: Corresponds to Table 6 of WO2021 / 110848. Selected mutations in novel SHC variant enzymes.

[0374]

[0375]

[0376] Note: These mutations are in addition to the mutations present in 215G2 SHC (M132R, A224V, and I432T). SEQ ID NOs: 2, 3, 4, 5, 17, and 18 in Table 8 correspond to SEQ ID NOs: 379, 2, 380, 381, 382, ​​and 383.

[0377] The sequences identified in Tables 7 and 8 above are represented by any one of SEQ ID NOs: 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 in the sequence listing and as shown at the end of the Examples section.

[0378] In some implementations, with SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 0, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 ,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、13 9, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 , 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 23 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 2 62, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,The SHC / HAC enzyme variant may have equal to or less than about 30 amino acid changes compared to SEQ ID NO: 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. For example, compared to SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63 ,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,9 7, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、1 99, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249,250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 311, 312, 313, 314, 315 The SHC / HAC enzyme variant can have equal to or less than about 25, or equal to or less than about 20, or equal to or less than about 15, or equal to or less than about 10, or equal to or less than about 9, or equal to or less than about 8, or equal to or less than about 7, or equal to or less than about 6 amino acid changes compared to SEQ ID NO: 9, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. For example, compared to SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,3 3, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 3, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 2 26, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 26 3, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310 Compared to 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the SHC / HAC enzyme variants may have at least about 5 or at least about 6 amino acid changes. These amino acid changes may be, for example, insertions, deletions, and / or substitutions as described above.

[0379] In some implementations, with SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 0, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 ,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、13 9, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 1 70, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 , 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 23 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 2 62, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,Compared to 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the only amino acid change in the SHC / HAC enzyme variants is substitution (i.e., no insertion or deletion).

[0380] Amino acid changes are defined relative to a reference sequence. An amino acid change relative to a reference sequence means that the amino acid sequence of the variant sequence differs from the reference sequence.

[0381] Amino acids in the reference sequence and variant sequences can be assigned a number, starting with the N-terminal amino acid of the polypeptide (i.e., the N-terminal amino acid of the polypeptide is numbered 1, the next amino acid is numbered 2, and so on). The "position" of the reference sequence refers to the specific amino acid residue present in the reference sequence, as determined by the specific numbering of the amino acid in the reference sequence. The "position" of the variant sequence refers to the specific amino acid residue present in the variant sequence, as determined by the specific numbering of the amino acid in the variant sequence.

[0382] Because a variant sequence may contain deletions or insertions compared to a reference sequence, the numbering of amino acids in the variant sequence may differ from the numbering of the same amino acid in the reference sequence. For example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO:1, the amino acid following the inserted amino acid will be numbered 133 in the variant sequence, while number 132 will remain in the reference sequence. In this example, the position in the variant sequence corresponding to position 132 in the reference sequence is position 133. Therefore, the amino acid retained from the reference sequence in the variant sequence can be defined by reference to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence can be defined by reference to the "corresponding position" in the reference sequence. Specifically, a substitution in a variant sequence compared to a reference sequence can be defined by reference to the "corresponding position" in the reference sequence, regardless of whether there are any insertions and / or deletions in the reference sequence. When an amino acid in the reference sequence has been deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions compared to the reference sequence (i.e., only substitutions), the "corresponding position" in the reference sequence will be the same as the position in the variant sequence.

[0383] For example, position 169 in AacSHC represented by SEQ ID NO: 1 corresponds to position 222 in ZmoSHC1 (SEQ ID NO: 13 or 14), position 177 in ZmoSHC2 (SEQ ID NO: 15), position 172 in TelSHC (SEQ ID NO: 23), and position 196 in ScoSH1 (SEQ ID NO: 32).

[0384] For example, position 306 in AacSHC represented by SEQ ID NO: 1 corresponds to position 368 in ZmoSHC1 (SEQ ID NO: 13 or 14), position 321 in ZmoSHC2 (SEQ ID NO: 15), position 311 in TelSHC (SEQ ID NO: 23), and position 335 in ScoSH1 (SEQ ID NO: 32).

[0385] For example, position 600 in AacSHC represented by SEQ ID NO: 1 corresponds to position 667 in ZmoSHC1 (SEQ ID NO: 13 or 14), position 619 in ZmoSHC2 (SEQ ID NO: 15), position 609 in TelSHC (SEQ ID NO: 23), and position 629 in ScoSH1 (SEQ ID NO: 32).

[0386] In one embodiment, the SHC / HAC enzyme variant has the same characteristics as SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 2 29, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and...

[0387] Where relative to SEQ ID NO: 1, 2, 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 8 4, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 13 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 16 5, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 1 96, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 2 27, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, the SHC / HAC enzyme variant amino acid sequence has an amino acid change at a position corresponding to position W169, A306 and / or G600 of SEQ ID NOs: 1, 2, 3, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NOs: 1, 2, 3 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0388] Compared to SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 1 09, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 1 40, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 1 71, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid alteration at positions W169, A306, and / or G600 corresponding to SEQ ID NO: 1, 2, or 3, or at positions W169, A306, and / or G600 corresponding to SEQ ID NO: 1, 2, or 3 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0389] In one embodiment, the SHC / HAC enzyme or enzyme variant (also known as type 1 variant) has the same characteristics as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54 and / or SEQ ID NO:55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 9 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 32, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 16 5, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 24 9, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 3 10, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55%. Amino acid sequences that are 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or similar to SEQ ID NO. ID NO:1, 2, or 3, with amino acid changes at the following positions: positions W169, A306, and / or G600 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, and / or 54, or positions such as SEQ ID NO:13, SEQ ID NO:14, corresponding to W169, A306, and / or G600 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, and / or 54.SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ IDNO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0390] In a preferred embodiment, the SHC / HAC enzyme or enzyme variant has at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 6% of the enzyme content of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. Amino acid sequences that are 7%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical or similar to the amino acid sequence, and have amino acid changes at positions W169, A306, and / or G600 corresponding to SEQ ID NO:1, 2, and 3, relative to SEQ ID NO:1, 2, and 3.

[0391] In one embodiment, the SHC / HAC enzyme variant (also known as type 2 variant) has the same SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 10 1, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 having an amino acid sequence that is at least 30.0% identical or similar to, and, relative to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, having an amino acid change at position corresponding to position W169 of SEQ ID NO: 1, 2, 3, or, corresponding to position W169 of SEQ ID NO: 1, 2, 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56 ,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 12 3, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 5, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53 or 54 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0392] In this case, the identity or similarity can be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0393] In one embodiment, the SHC / HAC enzyme variant (also referred to as type 3 variant) has the same NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 10 1, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 having an amino acid sequence that is at least 30.0% identical or similar to, and, relative to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, having an amino acid change at the following positions: W169 and G600M corresponding to positions of SEQ ID NO: 1, 2, 3, or W169 and G600M corresponding to SEQ ID NO: 1, 2, 3. NO:13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 8, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0394] In this case, the similarity or identity can be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, or 63%. 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0395] In one embodiment, a SHC / HAC enzyme variant (also referred to as a Type 4 variant) has

[0396] SEQ ID NO:1、2、3、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and, relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, having an amino acid change at the following positions: corresponding to positions W169, A306, and G600 of SEQ ID NO:1, 2, or 3, or corresponding to W169, A306, and G600 of SEQ ID NO:1, 2, or 3, as in SEQ ID NO. NO: 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 12 5, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 4, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0399] In this case, the identity or similarity may be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0400] All combinations of positional alterations in the above-mentioned SHC / AHC enzyme variants are included in this disclosure:

[0401] -W169

[0402] -W169, A306

[0403] -W169, A306, G600

[0404] -W169, G600

[0405] -A306, G600

[0406] -A306

[0407] -G600

[0408] (When referring to SEQ ID NO: 1).

[0409] The novel amino acid (X) at position 169 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 can be G, A, or V, or their functional equivalents. The functional equivalent of G is P. A and V are functional equivalents. M, L, and I are also functional equivalents of A and V. The preferred novel amino acid located at the following positions is G, A, or V: position 169 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or the position corresponding to position 169 of SEQ ID NO: 1, 2, or 3, as in SEQ ID NO: 1. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 1 62, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 25 The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in 3, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. The most preferred novel amino acid located at the following position is G: position 169 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or the position corresponding to position 169 of SEQ ID NO: 1, 2, or 3, as in SEQ ID NO: 1. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63 ,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,9 5, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 22 5, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 2 The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in numbers 66, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0410] For example, bit 169 of AacSHC represented by SEQ ID NO:1 corresponds to bit 222 of ZmoSHC1 (SEQ ID NO:13), bit 177 of ZmoSHC2 (SEQ ID NO:15), bit 172 of TelSHC (SEQ ID NO:23), and bit 196 of ScoSH1 (SEQ ID NO:32).

[0411] The novel amino acid (X) at position 306 of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 may be V or its functional equivalent. Functional equivalents of V are A, M, L, and I. Preferably, the novel amino acid located at position 306 of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, or 54 is V, or a position 306 of SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, 3. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in numbers 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0412] For example, the 306th bit in AacSHC represented by SEQ ID NO:1 corresponds to the 368th bit in ZmoSHC1 (SEQ ID NO:13), the 321st bit in ZmoSHC2 (SEQ ID NO:15), the 311th bit in TelSHC (SEQ ID NO:23), and the 335th bit in ScoSH1 (SEQ ID NO:32).

[0413] The novel amino acid (X) at position 600 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 may be A, V, L, I, or M, or a functional equivalent thereof. All of these amino acids are functional equivalents. Preferred novel amino acids located at positions 600 of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54 are A, V, L, I, or M, or positions 600 of SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, or positions 600 of SEQ ID NO:1, 2, or 3. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,6 1, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 7, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 26 The positions in the amino acid sequences of wild-type SHC / HAC enzymes shown in 6, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. The most preferred novel amino acid located at the following positions is M: position 600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or a position 600 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 7 2, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 9 2, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 10 9, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 2 04, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 23 8, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272 The positions of the amino acid sequences of wild-type SHC / HAC enzymes shown in 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0414] For example, bit 600 in AacSHC represented by SEQ ID NO:1 corresponds to bit 667 in ZmoSHC1 (SEQ ID NO:13), bit 619 in ZmoSHC2 (SEQ ID NO:15), bit 609 in TelSHC (SEQ ID NO:23), and bit 629 in ScoSH1 (SEQ ID NO:32).

[0415] Table 9: Positions of three preferred mutant amino acids (AA) in four different wild-type SHC / HACs

[0416]

[0417] In one embodiment, the SHC / HAC enzyme variant (also known as the type 5 variant) has the same SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 , 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 , 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and, relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, having an amino acid change at the following positions: corresponding to positions W169, A306, and / or G600 of SEQ ID NO:1, 2, or 3, or corresponding to W169, A306, and G600 of SEQ ID NO:1, 2, or 3 as in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 16 1, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 25 4. The positions of the amino acid sequences of wild-type SHC / HAC as shown in 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and the amino acid changes are:

[0418] - The W at position 169 is replaced by G, A, or V or their functional equivalent, preferably, wherein the W at position 169 is replaced by G.

[0419] - At position 306, A is replaced by V or its functional equivalent, and / or

[0420] - G at position 600 is substituted by A, V, L, I or M or a functional equivalent thereof, preferably, wherein G at position 600 is substituted by M.

[0421] In this case, the identity or similarity may be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0422] In one embodiment, the SHC / HAC enzyme variant (also known as the type 6 variant) has the same SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 , 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 , 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 have an amino acid sequence with at least 30.0% identity or similarity, and have an amino acid change at the following positions relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383: corresponding to position W169 of SEQ ID NO:1, 2, or 3, or corresponding to W169 of SEQ ID NO:1, 2, or 3 as in SEQ IDNO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 2 The positions in the amino acid sequence of the wild-type SHC / HAC enzyme shown in 70, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, where the amino acid change is the substitution of W at position 169 with G.

[0423] In one implementation, the 'W' in position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'G'.

[0424] In this case, the identity or similarity may be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0425] In one embodiment, the SHC / HAC enzyme variant (also known as the type 7 variant) has the same SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 , 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 , 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and, relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, having an amino acid change at the following positions: corresponding to positions W169 and G600M of SEQ ID NO:1, 2, or 3, or corresponding to W169 and G600M of SEQ ID NO:1, 2, or 3 as in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 12 5, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 The positions of the amino acid sequences of the wild-type SHC / HAC enzymes shown in 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and the amino acid changes are:

[0426] - The 169th position, W, was replaced by G, and

[0427] - The 600th letter G was replaced by M.

[0428] In one implementation, the 'W' in position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'G', and SEQ ID In the sequence NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the 600th G is replaced by M.

[0429] In this case, the similarity or identity can be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, or 63%. 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0430] In one embodiment, the SHC / HAC enzyme variant has the same characteristics as SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 7 0, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 2 29, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and, relative to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, with amino acid changes at the following positions:

[0431] The positions W169 and A306 corresponding to SEQ ID NO:1, 2, or 3, or the positions W169 and A306 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1. NO: 13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,The positions in the amino acid sequences of the wild-type SHC / HAC enzymes shown in 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and the amino acid changes are:

[0432] - The 169th position, W, was replaced by G, and

[0433] - A at position 306 is replaced by V.

[0434] In one implementation, the 'W' in position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'G', and

[0435] The 'A' in the 306th position of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'V'.

[0436] In this case, the similarity or identity can be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, or 63%. 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0437] In one embodiment, the SHC / HAC enzyme variant (also known as the type 8 variant) has the same SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 have an amino acid sequence with at least 30.0% identity or similarity, and, relative to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 having an amino acid change at the following positions: positions W169, A306 and G600 corresponding to SEQ ID NO: 1, 2, 3, or corresponding to W169, A306 and G600 of SEQ ID NO: 1, 2 or 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 12 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 1 62, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 The positions of the amino acid sequences of the wild-type SHC / HAC enzymes shown in 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid changes are:

[0438] - The 169th letter, W, was replaced by G.

[0439] - The 306th letter A was replaced by V, and

[0440] - The 600th letter G was replaced by M.

[0441] In one implementation, the 'W' in position 169 of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by 'G'.

[0442] -In SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, the 306th bit A is replaced by V, and

[0443] The 600th position of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 has its G replaced by M.

[0444] In this case, the identity or similarity may be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0445] In one embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and

[0446] Relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, in the corresponding SEQ ID The amino acid changes at positions W169 and G600 of NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 are as follows:

[0447] - W at position 169 is replaced by G, and

[0448] - G at position 600 is replaced by M.

[0449] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0450] In one embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and

[0451] Relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, in the corresponding SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 has an amino acid change at position W169 and A306, and the amino acid change is:

[0452] - W at position 169 is replaced by G, and

[0453] - A at position 306 is replaced by V.

[0454] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0455] In one embodiment, the SHC / HAC enzyme variant (also referred to as a Type 9 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and

[0456] With respect to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 has an amino acid change at position W169, A306 and / or G600, and the amino acid change is:

[0457] - The 169th letter, W, was replaced by G.

[0458] - The 306th letter A was replaced by V, and

[0459] - The 600th letter G was replaced by M.

[0460] In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0461] In a preferred embodiment, the SHC / HAC enzyme variant (also known as variant type 10) has an amino acid sequence that has at least 70.0% identity or similarity to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 305 or 306, and, relative to SEQ ID NO: The SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 305 or 306 have an amino acid change at position W169 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53 or 54, and the amino acid change is that W at position 169 is replaced by G.

[0462] In one embodiment, each SHC / HAC enzyme variant (Type 10 variant) has at least 70.0% identity or similarity to SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 53, 54, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 305 or 306, yet has the same mutations that make it different from SEQ ID NO: 1. Each of said mutations is defined herein, at least in the section entitled "Sequence Summary." In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0463] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P1 variant, SEQ ID NO: 312) (also referred to as Type 10 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 13 or 14, and has an amino acid alteration at position W222 of SEQ ID NO: 13 or 14 relative to SEQ ID NO: 13, and the amino acid alteration is a substitution of W at position 222 with G.

[0464] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:312, and still possesses W222G.

[0465] In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0466] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P2 variant: SEQ ID NO: 353) (also referred to as a Type 10 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 13, and has amino acid changes relative to SEQ ID NO: 13 at positions W222 and G667 of SEQ ID NO: 13, wherein the W at position 222 is substituted by G and the G at position 667 is substituted by M.

[0467] In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0468] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 13 and has amino acid changes relative to SEQ ID NO: 13 at positions W222 and A368 of SEQ ID NO: 13, wherein the amino acid changes are a substitution of W at position 222 with a G and a substitution of A at position 368 with a V.

[0469] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0470] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P3 variant: SEQ ID NO:354) (also known as variant 10) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:13, and has amino acid changes at positions W222, A368, and G667 relative to SEQ ID NO:13, wherein the amino acid changes are W at position 222 being replaced by G, A at position 368 being replaced by V, and G at position 667 being replaced by M.

[0471] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0472] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC2 P1 variant, SEQ ID NO:313) (also known as variant 10) has an amino acid sequence that is at least 70.0% identical or similar to that of SEQ ID NO:15, and has an amino acid change at position W177 of SEQ ID NO:15 relative to SEQ ID NO:15, wherein the amino acid change is that W at position 177 is replaced by G.

[0473] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:313, and still possesses W177G.

[0474] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0475] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC2 P2 variant: SEQ ID NO:355) (also known as variant 10) has an amino acid sequence that is at least 70.0% identical or similar to that of SEQ ID NO:15, and has an amino acid change at positions W177 and G619 relative to SEQ ID NO:15, wherein the amino acid change is that W at position 177 is replaced by G and G at position 619 is replaced by M.

[0476] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:313 or 355, and still possesses W177G and G619M.

[0477] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0478] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:15, and has an amino acid change at positions W177 and 321 of SEQ ID NO:15 relative to SEQ ID NO:15, wherein the amino acid change is that W at position 177 is replaced by G and A at position 321 is replaced by V.

[0479] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:313, and still possesses W177G and A321V.

[0480] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0481] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC2 P3 variant, SEQ ID NO: 356) (also referred to as Type 10 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 15, and has amino acid alterations relative to SEQ ID NO: 15 at positions W177, A321, and G619 of SEQ ID NO: 15, wherein the amino acid alterations are a substitution of W at position 177 with G, a substitution of A at position 321 with V, and a substitution of G at position 619 with M.

[0482] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:313, and still has W177G, A321V, and G619M.

[0483] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0484] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P1 SEQ ID NO:304) (also known as variant type 10) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:23, and has an amino acid change at position W172 of SEQ ID NO:23 relative to SEQ ID NO:23, wherein the amino acid change is that W at position 172 is replaced by G.

[0485] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:304, and still possesses W172G.

[0486] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:302, and still possesses W172G.

[0487] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0488] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P2 SEQ ID NO:359) (also known as variant type 10) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:23, and has amino acid changes at positions W172 and G609 relative to SEQ ID NO:23, wherein the amino acid changes are W at position 172 being replaced by G and G at position 609 being replaced by M.

[0489] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:359, and still possesses W172G and G609M.

[0490] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0491] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:23, and

[0492] Compared to SEQ ID NO:23, there are amino acid changes at positions W172 and A311 in SEQ ID NO:23, and the amino acid changes are that W at position 172 is replaced by G and A at position 311 is replaced by V.

[0493] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:360, and still possesses W172G and A311V.

[0494] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0495] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P3 SEQ ID NO:360) (also known as variant type 10) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:23, and

[0496] Relative to SEQ ID NO: 23, there are amino acid changes at positions W172, A311, and G609 of SEQ ID NO: 23, and the amino acid changes are substitution of W at position 172 by G, substitution of A at position 311 by V, and substitution of G at position 609 by M.

[0497] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:360, and still possesses W172G, A311V, and G609M.

[0498] In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0499] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P1 variant SEQ ID NO:311) (also referred to as Type 10 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:32, and has an amino acid alteration relative to SEQ ID NO:32 at position W196 of SEQ ID NO:32, wherein the amino acid alteration is a substitution of W at position 196 with G.

[0500] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:311, and still possesses W196G.

[0501] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P2 variant SEQ ID NO: 357) (also referred to as a Type 10 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 32 and has amino acid alterations relative to SEQ ID NO: 32 at positions W196 and G629 of SEQ ID NO: 32, wherein the amino acid alterations are a substitution of W at position 196 with G and a substitution of G at position 629 with M.

[0502] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO: 311, and still has W196G and G629M.

[0503] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:32, and has an amino acid change at positions W196 and A335 relative to SEQ ID NO:32, wherein the amino acid change is that W at position 196 is replaced by G and A at position 335 is replaced by V.

[0504] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO:311, and still possessing W196G and A335V. In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0505] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P3 variant SEQ ID NO:358) (also known as variant 10) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO:32, and has amino acid changes at positions W196, A335, and G629 relative to SEQ ID NO:32, wherein the amino acid changes are W at position 196 being replaced by G, A at position 335 being replaced by V, and G at position 629 being replaced by M.

[0506] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence having at least 70% identity or similarity to SEQ ID NO: 311, and still has W196G, A335V and G629M.

[0507] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0508] In a preferred embodiment, the enzyme variant is as follows:

[0509] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 1, 4, 5, 6 or 350, and has at least one of the following mutations: W169G, A306V and G600M, or

[0510] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 90.0% identical to SEQ ID NO: 7, 8, 9 or 386, and has the following mutations: M132R, A224V, I432T, A557T, R613S, and has at least one of the following mutations: W169G, A306V and G600M, or

[0511] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 90.0% identical to SEQ ID NO: 10, 11, 12 or 385, and has the following mutations: M132R, A224V, I432T, and has at least one of the following mutations: W169G, A306V and G600M, or

[0512] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and has at least one of the following mutations: W172G, A311V and G609M (Tel SHC / HAC variant), or

[0513] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has the following mutations: W196G, A335V and G629M (Sco SHC / HAC variant), or

[0514] - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353 or 354, and has at least one of the following mutations: W222G, A368V and G667M (Zmo SHC1 variant), or

[0515] - the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has at least one of the following mutations: W177G, A321V and G619M (Zmo SHC2 variant), or

[0516] - The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO:1 and has at least one of the following mutations: W169G, G600M, M132R, and I432T, or

[0517] - The SHC / HAC enzyme variant has at least 90.0% identity with SEQ ID NO:1 and has the following mutations: W169G, G600M, M132R and I432T, or

[0518] - The SHC / HAC enzyme variant has an amino acid sequence with at least 95.0% identity to SEQ ID NO:1 and has the following mutations: W169G, G600M, M132R, and I432T, or

[0519] - The SHC / HAC enzyme variant has an amino acid sequence that is at least 96.0%, 97.0%, 99.0%, and 99.0% identical to SEQ ID NO:1, and has the following mutations: W169G, G600M, M132R, and I432T.

[0520] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0521] In preferred embodiments, the SHC / HAC enzyme variant (also referred to as a Type 11 variant) has an amino acid sequence that is at least 70.0% identical or similar to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and, relative to SEQ ID NO: NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, in the sequence corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53 or 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 has an amino acid change at position W169 and G600M, and the amino acid change is:

[0522] - The 169th position, W, was replaced by G, and

[0523] - The 600th letter G was replaced by M.

[0524] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0525] In one embodiment, the SHC / HAC enzyme variant (also referred to as type 12 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 1 and has at least one of the following mutations relative to SEQ ID NO: 1: W169G, A306V, and G600M.

[0526] In one embodiment, the SHC / HAC enzyme variant (also referred to as type 13 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 4 and has at least the following mutation relative to SEQ ID NO: 1: W169G.

[0527] In one embodiment, the SHC / HAC enzyme variant (also known as the type 14 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:5 and has at least the following mutations relative to SEQ ID NO:1: W169G and G600M.

[0528] In one embodiment, the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:350, and has at least the following mutations relative to SEQ ID NO:1: W169G and A306V.

[0529] In one embodiment, the SHC / HAC enzyme variant (also known as the type 15 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:6 and has at least the following mutations relative to SEQ ID NO:1: W169G, A306V, and G600M.

[0530] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0531] In one embodiment, the SHC / HAC enzyme variant (also known as the type 16 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:3, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and at least one of the following mutations relative to SEQ ID NO:3: W169G, A306V, and G600M.

[0532] In one embodiment, the SHC / HAC enzyme variant (also known as the type 17 variant) has an amino acid sequence that is at least 70.0% identical to that of SEQ ID NO:10, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and the following mutation relative to SEQ ID NO:3: W169G.

[0533] In one embodiment, the SHC / HAC enzyme variant (also known as the type 18 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:11, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and the following mutations relative to SEQ ID NO:3: W169G and G600M.

[0534] In one embodiment, the SHC / HAC enzyme variant (also known as the type 32 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:385, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and the following mutations relative to SEQ ID NO:3: W169G and A306V.

[0535] In one embodiment, the SHC / HAC enzyme variant (also known as the type 19 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:12, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and the following mutations relative to SEQ ID NO:3: W169G, A306V, and G600M.

[0536] In this case, the similarity or identity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0537] In one embodiment, the SHC / HAC enzyme variant (also known as variant type 20) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:2, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and at least one of the following mutations relative to SEQ ID NO:2: W169G, A306V, and G600M.

[0538] In one embodiment, the SHC / HAC enzyme variant (also known as variant 21) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO:7, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and the following mutation relative to SEQ ID NO:2: W169G.

[0539] In one embodiment, the SHC / HAC enzyme variant (also referred to as a type 22 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 8, has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, A557T, R613S, and has the following mutations relative to SEQ ID NO: 2: W169G and G600M.

[0540] In one embodiment, the SHC / HAC enzyme variant (also referred to as a type 33 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 386, has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, A557T, R613S, and has the following mutations relative to SEQ ID NO: 2: W169G and A306V.

[0541] In one embodiment, the SHC / HAC enzyme variant (also referred to as a type 23 variant) has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 9, has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, A557T, R613S, and has the following mutations relative to SEQ ID NO: 2: W169G, A306V, and G600M.

[0542] In this case, the identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0543] In one embodiment, the SHC / HAC enzyme variant (also referred to as a Type 34 variant) has an amino acid sequence that is at least 30.0% identical or similar to SEQ ID NOs: 1-12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47-54, 55-296, 301-313, 315-353, 354-383, wherein W at position 169, or corresponding to position 169 of SEQ ID NO: 1, is substituted with G; wherein A at position 306, or corresponding to position 306 of SEQ ID NO: 1, 2 or 3, is substituted with V; and / or wherein G at position 600, or corresponding to position 600 of SEQ ID NO: 1, 2 or 3, is substituted with M.

[0544] In this case, the identity or similarity may be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, , 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0545] In one embodiment, the SHC / AHC enzyme variant (also known as variant 24) has an amino acid sequence comprising or consisting of or substantially consisting of the following: SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, 12, 304, 311, 312, 313, 305, 306, 302, 359, 357, 353, 355, 360, 358, 354, 356.

[0546] In one embodiment, the SHC / AHC enzyme variant (also known as variant 25) has an amino acid sequence comprising or consisting of or substantially consisting of the following: SEQ ID NO: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 350, 351, 352.

[0547] In one embodiment, each of the SHC / HAC enzyme variants described herein (type 1-34 variants) may have the following additional amino acid changes, wherein the (new) amino acid (X) may be S or a functional equivalent thereof at the position corresponding to position 168 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383. The functional equivalent of S is C, T, N or Q. at position 168 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, or at a position corresponding to position 168 of SEQ ID NO: 1, 2 or 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 5 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 2, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 1 59, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 At the positions shown in the amino acid sequences of wild-type SHC / HAC as indicated by 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, the preferred new amino acid is S.

[0548] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and

[0549] - Relative to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53 and / or 54, there is an amino acid change at the following positions: position W169 corresponding to SEQ ID NO:1, 2, 3, or position W169 corresponding to SEQ ID NO:1, 2, 3 as in SEQ ID NO:1, 2, 3. NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,64,65,66,67,68 ,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 of the amino acid sequence of the wild-type SHC / HAC enzyme, and the amino acid change is that W at position 169 is substituted by G, and

[0550] - relative to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, having an amino acid S at position 168 and at the following positions: position S168 corresponding to S168 of SEQ ID NO: 1, 2, 3, or a residue corresponding to S168 of SEQ ID NO: 1, 2, 3 or a residue corresponding to S168 of SEQ ID NO: 1, 2, 3 or a residue corresponding to S168 of SEQ ID NO: NO:13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,55,56,57,58,59,60,61,62,63,6 4, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 1 95, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0551] In this case, the similarity or identity can be at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, or 63%. 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0552] In one embodiment, by referring to the wild-type Aac SHC / HAC having SEQ ID NO:1 or any variant derived from SEQ ID NO:1 and having SEQ ID NO:2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, any SHC / HAC enzyme variant may contain at least one of the following conserved amino acids: L22, Q26. , G30, W32, A44, L48, Q72, G76, W78, Y95, L98, G102, A113, I117, G121, G122, F129, T130, L134, A135, G 138, W142, P146, W169, A170, R171, F217, D222, R237, I261, P263, P281, S309, P310, W312, D313, T314, A320, W339, Q344, G349, D350, W351, G361, G362, A364, F365, N369, Y372, P373, D374, D376, D377, W40 6. Q411, G415, A419, P433, D436, D442, P443, D447, V448, Q479, G483, W485, G487, R488, W489, G490, N4 92, Y495, G496, T497, L504, W522, Q527, G531, G532, W533, G534, E535, S539, Y540, G547, T552, T556, W558, A559, A565, L581, Q585, G589, W591, G600, F601, P602, F605, Y609, Y612, F616, P617, A620 and R623. The variants may also be derived from any wild-type SHC / HAC enzyme as defined herein (e.g., SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、2 56, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), wherein the confirmed corresponding position corresponds to the position confirmed as described above for SEQ ID NO:1. The variants may be derived from any variant type defined above herein.

[0553] Thus, provided herein is a method for preparing (-)-Ambroxol by enzymatically converting EEH to (-)-Ambroxol. Also provided herein is a method for preparing Ambroxol by enzymatically converting E,E-Dihomofarnesol to Ambroxol. The method can use any of the SHC / HAC enzyme variants described herein (particularly variants 1 to 25).

[0554] "Percent (%) identity" with respect to polypeptide or nucleotide sequences is defined as the percentage of amino acids or nucleotides in the candidate sequence that are identical to the amino acids or nucleotides in the reference sequence, respectively, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences. The terms "polypeptide" and "protein" are used interchangeably herein to refer to any amino acid linked peptide chain, regardless of its length or post-translational modifications.

[0555] The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined by sequence alignment. Such comparisons can be carried out using a variety of algorithms known in the art, preferably using the mathematical algorithm (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877) of Karlin and Altschul, using hmmalign (HMMER bag, http: / / hmmer.wustl.edu / ) or using CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), available from, for example: https: / / www.ebi.ac.uk / Tools / msa / clustalo / or GAP program (mathematical algorithm of the University of Iowa) or the mathematical algorithm (1989-Cabios 4: 11-17) of Myers and Miller. The preferred parameters used are the default parameters, as they are set at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0556] Percent sequence identity can be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches can be performed using the BLASTN program with a score of 100 and a word length of 12 to obtain polynucleotide sequences homologous to nucleic acids encoding related proteins. BLAST protein searches can be performed using the BLASTP program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to polypeptides.

[0557] To obtain gap alignments for comparative purposes, gap BLAST can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using BLAST and gap BLAST procedures, the default parameters of the respective procedures should be used. Sequence matching analysis can be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are mentioned in this application, unless otherwise explicitly stated, these percentages are calculated relative to the full length of longer sequences.

[0558] In a specific embodiment, the % identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0559] Additional amino acid changes may be present in the SHC / AHC variants disclosed herein. In one embodiment, an example of an additional amino acid change is shown relative to SEQ ID. NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 1 36, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310, and at the following position: corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 position 81, 431, 90, 172, 277, 37, 174, 601, 77, 92, 129, 579, 605, or a sequence corresponding to position 81, 431, 90, 272, 177, 37, 174, 601, 77, 92, 129, 579, 605 of SEQ ID NO:1, 2 or 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 3 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 6, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 24 5, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0560] Type 26 variants: In one embodiment, additional amino acid changes are exemplified relative to SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 3 66, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 3 81, 382, ​​383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,1 39, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and at the following positions: corresponding to SEQ ID Positions 81 and / or 431 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or positions 81 and 431 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58 ,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 12 9, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258 8, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0561] 27 variants: In one embodiment, additional amino acid changes are exemplified relative to SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,1 39, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and at the following positions: corresponding to SEQ ID Positions 90, 172, and / or 277 of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or positions 90, 272, or 177 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3. NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 5 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 12 8, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 1 68, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 7, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0562] Type 28 variant: In one embodiment, an example of additional amino acid changes relative to SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,1 39, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and at the following positions: corresponding to SEQ ID Positions 37, 174, and 601 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or positions 37, 174, and 601 corresponding to SEQ ID NO:1, 2, or 3. NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 38 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 5, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 The positions in the amino acid sequences of wild-type SHC / HAC enzymes, as shown in numbers 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0563] Variant 29: In one embodiment, an example of additional amino acid changes relative to SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,1 39, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 9, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310, and at positions corresponding to positions 77, 92, 129, 579, 601, 605 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53 and / or 54, or corresponding to positions 77, 92, 129, 579, 601, 605 of SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, , 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0564] at a position corresponding to position 81 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 (or at a position corresponding to position 81 of SEQ ID NO: 1, 2 or 3 such as SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20 For example, the amino acid change at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in FIG. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be Y81X. This means that the amino acid Y at position 81 of SEQ ID NO: 1 is replaced by any different amino acid (X). As described above, since the SHC / HAC enzyme variant may further include insertions and / or deletions, the number of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 81.

[0565] At position 81 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.) The new amino acid (X) at the position (position 81) of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Phe. For example, an amino acid change at position 81 corresponding to SEQ ID NO:1 can replace the amino acid (i.e., Y) of SEQ ID NO:1 with a basic amino acid (i.e., His, Lys, or Arg). For example, at position 81 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., Y) in SEQ ID NO:1 with histidine (i.e., the amino acid change corresponding to position 81 in SEQ ID NO:1 is Y81H).

[0566] 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). The amino acid change at position 431 of NO: 1, 2, 3 (as shown in the amino acid sequence of the wild-type SHC / HAC enzyme as shown in 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, H431X. This refers to a position at position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 (or at a position corresponding to position 81 of SEQ ID NO: 1, 2 or 3). The amino acid H at position ( ) in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 431.

[0567] At position 431 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, 3, or SEQ ID NO:3) The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 431 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., H) in SEQ ID NO:1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile).For example, at position 431 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, 3, etc.). Amino acid changes at positions 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme can replace the amino acid (i.e., H) in SEQ ID NO:1 with leucine (i.e., the amino acid change corresponding to position 431 in SEQ ID NO:1 is H431L).

[0568] At position 90 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 90 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, T90X. This refers to position 90 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or in position 90 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid T at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is substituted with any different amino acid (X). As described above, since the SHC / HAC enzyme variant may further include insertions and / or deletions, the number of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 90.

[0569] at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380 The new amino acid (X) at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr or Phe. For example, at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 90 of SEQ ID NO: 1, 2, or 3). Amino acid changes at positions (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid of SEQ ID NO: 1 (i.e., T) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu or Ile).For example, at position 90 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 90 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., T) in SEQ ID NO:1 with alanine (i.e., the amino acid change corresponding to position 90 in SEQ ID NO:1 is T90A).

[0570] At position 172 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 172 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.) Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, A172X. This refers to position 172 in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 172 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1, 2, or 3). The amino acid T at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is substituted with any different amino acid (X). As described above, since the SHC / HAC enzyme variant may further include insertions and / or deletions, the number of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 172.

[0571] At position 172 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 172 corresponding to SEQ ID NO: 1, 2, or 3, as SEQ ID NO: 1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 172 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 172 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace amino acid A in SEQ ID NO:1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, Gln).For example, at position 172 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 172 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid change at positions 172 in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can replace the amino acid (i.e., A) in SEQ ID NO:1 with threonine (i.e., the amino acid change corresponding to position 172 in SEQ ID NO:1 is A172T).

[0572] At position 277 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 277 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, M277X. This refers to position 277 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 277 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:2). The amino acid M at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 277.

[0573] At position 277 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 81 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 277 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 277 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (M) in SEQ ID NO:1 with a basic amino acid (His, Lys, Arg).For example, at position 277 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 277 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid change at position (in the amino acid sequence of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., M) in SEQ ID NO:1 with lysine, that is, the amino acid change corresponding to position 277 in SEQ ID NO:1 is M277K.

[0574] At position 37 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 37 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, L37X. This refers to position 37 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 37 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:37). The amino acid L at positions (in the amino acid sequences of the wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 37.

[0575] At position 37 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 37 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 37 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 37 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) (or at position 37 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 38 Amino acid changes at positions 37, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 of the wild-type SHC / HAC enzyme can replace the amino acid L in SEQ ID NO: 1 with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln).For example, at a position corresponding to position 37 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 37 of SEQ ID NO: 1, 2, or 3). The amino acid change at the position corresponding to position 37 of SEQ ID NO: 1 is L37Q) in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can replace the amino acid of SEQ ID NO: 1 (i.e., L) with glutamine (i.e., the amino acid change at the position corresponding to position 37 of SEQ ID NO: 1 is L37Q).

[0576] At position 174 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 174 corresponding to SEQ ID NO: 1, 2, or 3, as SEQ ID NO: 1). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, V174X. This refers to position 174 in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 277 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1, 2, or 3). The amino acid V at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 174.

[0577] At position 174 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 174 corresponding to SEQ ID NO: 1, 2, or 3, as SEQ ID NO: 1). The new amino acid (X) at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Met, Ala, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr or Phe. at a position corresponding to position 174 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 174 of SEQ ID NO: 1, 2, or 3). The new amino acid (X) at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can, for example, be a hydrophobic amino acid (i.e., Met, Ala, Leu or Ile).For example, at position 174 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 174 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., V) in SEQ ID NO:1 with isoleucine (i.e., the amino acid corresponding to position 174 in SEQ ID NO:1 is replaced with V174I).

[0578] at a position corresponding to position 601 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 601 of SEQ ID NO: 1, 2, or 3). The amino acid change at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, F601X. This refers to a position at position 601 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 (or at a position corresponding to position 601 of SEQ ID NO: 1, 2 or 3). The F substitution at position ( ) in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 601.

[0579] at a position corresponding to position 601 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 601 of SEQ ID NO: 1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp or Tyr. At position 601 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 601 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1, 2, or 3). The new amino acid (X) at the positions shown in the amino acid sequences of wild-type SHC / HAC enzymes NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, an aromatic acid (i.e., Trp, Tyr, Phe).For example, at position 601 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 601 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid change at positions (in the amino acid sequence of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., F) in SEQ ID NO:1 with tyrosine, that is, the amino acid change corresponding to position 601 in SEQ ID NO:1 is F601Y).

[0580] At position 77 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 77 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, T77X. This refers to position 77 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 77 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid T at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 77.

[0581] At position 77 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 77 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The new amino acid (X) at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr or Phe. For example, at position 77 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 77 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid of SEQ ID NO: 1 (i.e., T) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu or Ile).For example, at a position corresponding to position 77 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 77 of SEQ ID NO: 1, 2, or 3). The amino acid change at the position corresponding to position 77 of SEQ ID NO: 1 is T77A).

[0582] At position 92 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 92 corresponding to SEQ ID NO:1, 2, 3, or SEQ ID NO:3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, I92X. This refers to position 92 in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 92 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid I at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is substituted with any different amino acid (X). As described above, since the SHC / HAC enzyme variant may further include insertions and / or deletions, the number of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 92.

[0583] At position 92 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 92 corresponding to SEQ ID NO:1, 2, or 3, as in SEQ ID NO:1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 92 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 92 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be used to replace the amino acid (i.e., I) of SEQ ID NO:1 with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile).For example, at position 92 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 92 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). The amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be used to replace the amino acid (i.e., I) of SEQ ID NO:1 with valine (i.e., the amino acid change corresponding to position 92 of SEQ ID NO:1 is I92V).

[0584] At position 129 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 129 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, F129X. This refers to position 129 in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or position 129 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1). The amino acid F at position (in the amino acid sequence of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 129.

[0585] At position 129 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 129 corresponding to SEQ ID NO: 1, 2, or 3, such as SEQ ID NO: 1, 2, or 3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp or Tyr. For example, at a position corresponding to position 129 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 129 of SEQ ID NO: 1, 2, or 3). Amino acid changes at positions (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid of SEQ ID NO: 1 (i.e., F) with a hydrophobic amino acid (i.e., Met, Ala, Val, Leu or Ile).For example, at position 129 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 129 corresponding to SEQ ID NO:1, 2, or 3, such as SEQ ID NO:1, 2, or 3). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (i.e., F) in SEQ ID NO:1 with leucine (i.e., the amino acid change corresponding to position 129 in SEQ ID NO:1 is F129L).

[0586] At position 579 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 579 corresponding to SEQ ID NO:1, 2, 3, or SEQ ID NO:3). The amino acid change at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Q579X. This refers to position 579 in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or in the position 579 corresponding to SEQ ID NO: 1, 2, 3, such as SEQ ID NO: 579). The amino acid Q at position (in the amino acid sequence of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is replaced with any different amino acid (X). As mentioned above, since SHC / HAC enzyme variants may additionally contain insertions and / or deletions, the number of the new amino acid (X) in a new SHC / HAC enzyme variant may not be 579.

[0587] At position 579 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 579 corresponding to SEQ ID NO:1, 2, 3, or SEQ ID NO:3). The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at position 579 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 579 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.). Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can replace the amino acid (Q) in SEQ ID NO:1 with a basic amino acid (His, Lys, or Arg).For example, at position 579 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 579 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.). The amino acid change at the positions shown in the amino acid sequences of wild-type SHC / HAC enzymes NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can replace the amino acid (i.e., Q) in SEQ ID NO:1 with histidine (i.e., the amino acid change corresponding to position 579 in SEQ ID NO:1 is Q579H).

[0588] At position 605 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 605 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.) Amino acid changes at positions (in the amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, F605X. This refers to position 605 in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or in position 605 corresponding to SEQ ID NO: 1, 2, 3, etc.). The amino acid F at position (in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) is substituted with any different amino acid (X). As described above, since the SHC / HAC enzyme variant may further include insertions and / or deletions, the number of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 605.

[0589] At position 605 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 605 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.) The new amino acid (X) at the position in the amino acid sequence of the wild-type SHC / HAC enzyme shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp or Tyr. At position 605 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at position 605 corresponding to SEQ ID NO:1, 2, 3, such as SEQ ID NO:1, 2, 3, etc.) The new amino acid (X) at the positions shown in the amino acid sequences of wild-type SHC / HAC enzymes NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 can be, for example, an aromatic acid (i.e., Trp, Tyr, Phe). For example, an amino acid change at position 605 corresponding to SEQ ID NO:1, 2, or 3 (or at a position in the amino acid sequence of wild-type SHC / HAC enzymes such as SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 corresponding to position 605 of SEQ ID NO:1) can replace the amino acid F in SEQ ID NO:1 with tryptophan (i.e., the amino acid change at position 601 corresponding to SEQ ID NO:1 is F605W).

[0590] For example, the SHC / HAC enzyme variants are located at positions 169, 306, 600, 132, and 432 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 132, and 432 corresponding to SEQ ID NO:1, 2, or 3, as shown in SEQ ID NO:3). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0591] For example, SHC / HAC enzyme variants, as previously defined herein, can be located at positions 169, 306, 600, 601 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 601 corresponding to SEQ ID NO:1, 2, or 3, as shown in SEQ ID NO:3). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0592] For example, SHC / HAC enzyme variants, as previously defined herein, can be located at positions 169, 306, 600, 77, 92, and 129 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 77, 92, and 129 corresponding to SEQ ID NO: 1, 2, or 3, as shown in SEQ ID NO: 1). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0593] For example, SHC / HAC enzyme variants as previously defined herein may be located at positions 169, 306, 600, 579, and 601 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 579, and 601 corresponding to SEQ ID NO:1, 2, or 3, as shown in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, as shown in SEQ ID NO:1, 2, or 3). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0594] For example, SHC / HAC enzyme variants, as previously defined herein, can be located at positions 169, 306, 600, 129, 132, and 432 corresponding to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 129, 132, and 432 corresponding to SEQ ID NO:1, 2, or 3, as shown in SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, as shown in SEQ ID NO:1, 2, 3, 432). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0595] For example, SHC / HAC enzyme variants, as previously defined herein, can be located at positions 169, 306, 600, 132, 432, and 601 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 132, 432, and 601 corresponding to SEQ ID NO: 1, 2, or 3, as shown in SEQ ID NO: 1). The amino acid sequences of wild-type SHC / HAC enzymes shown in NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0596] For example, SHC / HAC enzyme variants, as previously defined herein, can be located at positions 169, 306, 600, 129, 132, 432, and 601 corresponding to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at positions 169, 306, 600, 129, 132, 432, and 601 corresponding to SEQ ID NO: 1, 2, or 3, as in SEQ ID NO: 1, 2, 3, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 1, 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 1 ...1, SEQ ID NO: 3, SEQ ID NO: 1, The amino acid sequences of wild-type SHC / HAC enzymes shown in IDNOs 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 have amino acid alterations (e.g., substitutions).

[0597] For example, novel SHC / HAC enzyme variants can exhibit attractive biological activities as previously defined herein. Compared to the SHC / HAC enzymes of SEQ ID NO:1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296 or 307-310,...

Claims

1. A method for preparing (-)-Ambroxol or a mixture comprising (-)-Ambroxol, the method comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a homofarnesol isomer mixture comprising EEH into (-)-Ambroxol or a mixture comprising (-)-Ambroxol using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has the same amino acid sequence as SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,47,48,49,50,51,52,53,54,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382 or 383,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 2 22, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 2 64, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 having an amino acid sequence with at least 30.0% identity or similarity,and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid change at a position corresponding to position W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3 in the amino acid sequence of the wild-type SHC / HAC enzyme relative to the following sequence: NO:1,2,3,47,48,49,50,51,52,53,54,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382 or 383,55,56,57,58,59,60,61,62,63,64,65 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、 152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190,191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. , 2. A method for preparing (-)-ambroxol or a mixture comprising (-)-ambroxol, the method comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH into (-)-ambroxol or a mixture comprising (-)-ambroxol using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 13, 15, 23, 32, and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid change relative to SEQ ID NO: 1 at a position corresponding to position W169, A306 and / or G600 of SEQ ID NO: 1, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NO: 1 in the amino acid sequence of a wild-type SHC / HAC enzyme.

3. A method for preparing ambergris oxide or a mixture comprising ambergris oxide, the method comprising enzymatically converting (2,E)-dihomofarnesol (BisEEH) or a dihomofarnesol isomer mixture comprising BisEEH into ambergris oxide or a mixture comprising ambergris oxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has the same sequence as SEQ ID NO: NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,47,48,49,50,51,52,53,54,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382 or 383,55,56,57,58,59,60,61,62,63,64,65,66,67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220 , 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263 3, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 having an amino acid sequence with at least 30.0% identity or similarity,and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid change at a position corresponding to position W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NO: 1, 2 or 3 in the amino acid sequence of the wild-type SHC / HAC enzyme relative to the following sequence: NO:1,2,3,47,48,49,50,51,52,53,54,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382 or 383,55,56,57,58,59,60,61,62,63,64,65 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244 4, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. , 4. A method for preparing ambergris oxide or a mixture comprising ambergris oxide, the method comprising enzymatically converting (2,E)-dihomofarnesol (BisEEH) or a dihomofarnesol isomer mixture comprising BisEEH into ambergris oxide or a mixture comprising ambergris oxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 13, 15, 23, 32, and wherein the SHC / HAC enzyme variant amino acid sequence has an amino acid change relative to SEQ ID NO: 1 at a position corresponding to position W169, A306 and / or G600 of SEQ ID NO: 1, or at a position corresponding to W169, A306 and / or G600 of SEQ ID NO: 1 in the amino acid sequence of a wild-type SHC / HAC enzyme.

5. A method according to any one of the preceding claims, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted by G, A or V or a functional equivalent thereof, preferably wherein W at position 169 is substituted by G, - the A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted by V or a functional equivalent thereof, and / or - the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with A, V, L, I or M or a functional equivalent thereof.

6. A method according to any one of the preceding claims, wherein: - the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with G.

7. A method according to any one of the preceding claims, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with G, and - the A at position 306 in SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with V.

8. A method according to any one of the preceding claims, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with G, and - the G at position 600 in SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with M.

9. A method according to any one of the preceding claims, wherein: - the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with G, - the A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with V, and - the G at position 600 in SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is substituted with M.

10. The method of any of the preceding claims, wherein in SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 6, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,The amino acid at position 168 of 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 is S, or corresponds to the amino acid sequence of SEQ ID NO: 1 in the wild-type SHC. NO:1,2,3,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,47,48,49,50,51,52,53,54,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382 or 383,55,56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, The amino acid at position 168 of 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309 or 310 is S.

11. A method according to any one of the preceding claims, wherein - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 1, 4, 5, 6 or 350, and having at least one of the following mutations: W169G, A306V and G600M, or - an SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 7, 8, 9 or 386, and having the following mutations: M132R, A224V, I432T, A557T, R613S, and having at least one of the following mutations: W169G, A306V and G600M, or - The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 10, 11, 12 or 387 and has the following mutations: M132R, A224V, I432T and at least one of the following mutations: W169G, A306V and G600M.

12. The method according to any one of claims 1 to 11, wherein W at position 169 is substituted by G.

13. The method according to any one of claims 1 to 11, wherein W at position 169 is substituted by G, and A at position is substituted by V.

14. The method according to any one of claims 1 to 11, wherein W at position 169 is substituted by G, and G at position 600 is substituted by M.

15. The method according to any one of claims 1 to 11, wherein W at position 169 is substituted by G, A at position 306 is substituted by V, and G at position 600 is substituted by M.

16. A method according to any one of the preceding claims, wherein - a SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and having at least one of the following mutations: W172G, A311V and G609M (Tel SHC / HAC variant), or - a SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and having the following mutations: W196G, A335V and G629M (Sco SHC / HAC variant), or - a SHC / HAC enzyme variant having an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and having at least one of the following mutations: W222G, A368V and G667M (Zmo SHC1 variant), or - The SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has at least one of the following mutations: W177G, A321V and G619M (Zmo SHC2 variant).

17. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and has a W172G mutation.

18. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and has W172G and A311V mutations.

19. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and has W172G and G609M mutations.

20. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 23, 304, 359 or 360, and has W172G, A311V and G609M mutations.

21. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has a W196G mutation.

22. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has W196G and A335V mutations.

23. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has W196G and G629M mutations.

24. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence at least 70.0% identical to SEQ ID NO: 32, 311, 357 or 358, and has W196G, A335V and G629M mutations.

25. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and has a W222G mutation.

26. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and has W222G and A368V mutations.

27. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and has W222G and G667M mutations.

28. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 13, 312, 353 or 354, and has W222G, A321V and G667M mutations.

29. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has a W177G mutation.

30. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence that is at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has W177G and A321V mutations.

31. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has W177G and G619M mutations.

32. The method of any one of claims 1 to 16, wherein the SHC / HAC enzyme variant has an amino acid sequence at least 70.0% identical to SEQ ID NO: 15, 313, 355 or 356, and has W177G, A321V and G619M mutations.

33. The method according to any one of the preceding claims, wherein the SHC / AHC enzyme variant has an amino acid sequence comprising, consisting of, or consisting essentially of the following sequence: SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 305, 306, 304, 302, 311, 312, 313, 353, 354, 355, 356, 357, 358, 359, 360, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 350, 351, 352, 385 or 386.

34. A method according to any one of the preceding claims, wherein: The SHC / HAC enzyme variants disclosed herein exhibit: - Increased substrate specificity for EEH (or for BisEEH) when using homofarnesol or dihomofarnesol substrates, - when homofarnesol or dihomofarnesol substrates are used, the product selectivity towards (-)-ambroxol (or ambergris oxide) is increased, - when a substrate other than homofarnesol or dihomofarnesol is used (e.g. ethyl-homofarnesol, hydroxyfarnesylacetone, etc. - see, e.g. WO2021 / 110858 and WO2021 / 209482, respectively), the specificity for a particular isomer of the substrate is increased, - increased productivity, and / or - within the first 3 to 6 hours of the reaction (or within the first 4, 5 or 6 hours), the degree of conversion of EEH (or BisEEH) increases and the conversion rate of EEH (or BisEEH) increases, The increase is compared to a reference SHC enzyme (e.g., wild-type SHC, e.g., represented by SEQ ID NO: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 2, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206 06, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238 , 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 2 71, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or those indicated in 215G2. A parent SHC enzyme from which AacSHC or SHC#65 or a variant is derived (e.g., those represented by SEQ ID NO: 2, 3, 47-48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383)).

35. A SHC / HAC enzyme variant as defined in any one of the preceding claims.

36. The method according to any one of the preceding claims except claim 35, wherein the enzymatic conversion is carried out at a temperature range of about 30°C to about 50°C, such as a temperature range of about 40°C to about 50°C, and / or at a pH range of about 5 to about 6.

37. The method according to any one of the preceding claims except claim 35, wherein the method comprises culturing a recombinant host cell producing the SHC / HAC enzyme variant.

38. The method of claim 35, wherein the recombinant host cell comprises a nucleic acid sequence selected from SEQ ID NO: 38, 39, 40, 41, 42, 43, 44, 45 or 46.

39. The method according to any one of the preceding claims except claim 35, wherein the homofarnesol isomer mixture comprising EEH is an EE:EZ isomer mixture, preferably, wherein the weight ratio of the EE:EZ isomer mixture is 80:

20.

40. The method of any one of the preceding claims except claim 35, wherein the weight ratio of the recombinant host cell producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is about 2:1, 1:1, or about 0.5:1, or about 0.1:1, and the homofarnesol isomer mixture comprising EEH is an 80:20 weight ratio EE:EZ isomer mixture.

41. The method according to any one of the preceding claims except claim 35, wherein the weight ratio of the recombinant host cell producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is in the range of 0.1-0.5, preferably 0.4, and the concentration of EEH is in the range of 250 g / l to 650 g / l, preferably 450 g / l, and the mixture is an EE:EZ isomer mixture.

42. The method according to any one of the preceding claims except claim 35, wherein the weight ratio of the recombinant host cell producing the SHC / HAC enzyme variant to EEH or a homofarnesol isomer mixture comprising EEH is 1, and the concentration of EEH is 250 g / l, and the mixture is an EE:EZ isomer mixture.

43. (-)-Ambroxan obtained or obtainable by the process of any preceding claim except claim 35, in solid form in amorphous or crystalline form.

44. Use of (-)-Ambroxan as claimed in claim 43 as part of a fragrance or cosmetic or consumer product such as a fabric care, toiletries, beauty care, cleaning product, detergent product and / or soap product.

45. A fragrance or cosmetic or consumer product comprising the (-)-Ambroxan according to claim 43.

46. ​​A nucleic acid sequence encoding the SHC / HAC enzyme variant according to claim 35.

47. A construct comprising the nucleic acid sequence of claim 46.

48. A recombinant host cell comprising the nucleic acid sequence of claim 46 or the construct of claim 47.

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