Beta-amylase

Abstract

A variant polypeptide having beta-amylase activity, and the manufacture of the enzyme and the use of the enzyme in baking, detergents, personal care products, in fabric treatment, in pulp and paper treatment, in ethanol, lignocellulosic ethanol or syrup; the invention also relates to a method for using the enzyme as a viscosity reducer in the oil field and mining industry.

Description

[0001] This application is a divisional application of PCT application PCT / IB2018 / 001276, filed on October 17, 2018, with the invention name “β-amylase”. The date on which the PCT application entered the Chinese national phase is April 24, 2020, and the application number is 201880069776.5.

[0002] Sequence Listing

[0003] The present application includes a nucleotide and amino acid sequence listing in computer readable form (CRF) in an ASCII text (.txt) file according to "Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in International Patent Applications Under the Patent Cooperation Treaty (PCT)" ST.25. The sequence listing is identified below and is incorporated into the present specification of the present application in its entirety by reference and for all purposes.

[0004] file name Creation Date Size (bytes) 170325 sequence listing.txt October 10, 2018 29.3KB (30,022 bytes) Technical Field

[0005] Bread has been a staple food for human nutrition for thousands of years. Bread is usually made by combining flour, water, salt, yeast and / or other food additives to make dough or paste; the dough is then baked to make bread. Known enzymes are useful in baking because the effect of enzymes on the baking process may be similar or better than chemical substitutes. Several different enzymes can be used to make bread, for example, known amylases help to maintain freshness (anti-aging or hardness) over time and maintain elasticity over time. However, the baking industry still needs amylases that can provide fresh bread over a longer period of time than the existing time, or amylases that can provide bread that is better than fresh bread over time. A solution to this problem is a variant polypeptide with β-amylase activity that meets or exceeds these industrial requirements. In addition, β-amylases can be used in animal feed, detergents, personal care products, fabric treatment, pulp and paper treatment, in ethanol production, in lignocellulosic ethanol production, in syrup production, or as a viscosity reducer in the oil field and mining industry. Summary of the invention

[0006] A variant polypeptide having beta-amylase activity, wherein the variant polypeptide is encoded by a nucleic acid sequence that is at least 80% identical, 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%, at least 99% or at least 100% identical to the nucleic acid sequence set forth in SEQ ID NO:1.

[0007] A variant polypeptide having β-amylase activity, wherein the variant polypeptide is selected from the group consisting of: (a) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:2 at the following amino acid residue position number: at least one single amino acid modification of the amino acid sequence of NO:2: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:2 NO:2 and any combination thereof, and the variant polypeptide has beta-amylase activity; (b) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:3, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:4 at the following amino acid residue position: at least one single amino acid modification of the amino acid sequence of NO:3: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:3 Any combination of the amino acid sequence of IDNO: 3, and the variant polypeptide has β-amylase activity;(b) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:4, wherein the variant polypeptide has an amino acid sequence at the following amino acid residue position numbering to that of SEQ ID NO:4: at least one single amino acid modification of the amino acid sequence of NO:4: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:4 or any combination thereof, and the variant polypeptide has beta-amylase activity; (d) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:5, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:4 at the following amino acid residue position number: at least one single amino acid modification of the amino acid sequence of NO:5: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:5 Any combination of the amino acid sequence of IDNO: 5, and the variant polypeptide has β-amylase activity;(e) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:6, wherein the variant polypeptide has an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:6, at least one single amino acid modification of the amino acid sequence of NO:6: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:6, and the variant polypeptide has beta-amylase activity; and (f) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:7, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:7 at the following amino acid residue position numbering. at least one single amino acid modification of the amino acid sequence of NO:7: 48, 51, 56, 57, 59, 60, 80, 115, 133, 137, 139, 151, 173, 175, 177, 178, 179, 185, 187, 204, 207, 211, 229, 233, 235, 237, 238, 247, 251, 252, 253, 262, 305, 231, 318, 351, 354, 396, 398, 401, 430, 431, 470, 472, 478, 484, 485, 488, 490, 495, 496, 497, 500, 508, 522, 531, 536, 540, or to SEQ ID NO:7 Any combination of the amino acid sequence of NO:7, and the variant polypeptide has β-amylase activity. ;

[0008] The variant polypeptide, wherein the at least one single amino acid modification is an amino acid substitution, insertion, deletion or any combination thereof, and the variant polypeptide has β-amylase activity.

[0009] The variant polypeptide, wherein the amino acid substitution is a conservative amino acid substitution.

[0010] The variant polypeptide, wherein the at least one single amino acid modification is an amino acid substitution comprising the following: K16Q, D19I, D19L, K24D, K24E, D25P, L27Q, L27H, L27C, I28A, K48Q, E48D, D51I, D51L, K56D, K56E, D57P, D57K, L59Q, L59H, L59C, I60A, E80D, C83S, S83C, T101N, Q105L, N107S, C115S, S115C, N119D, T133N, Q137L, N139S, A141R, N143D, S145N, S146 P, Y147H, N151D, K153E, Y155H, H155Y, S172T, A173R, N175D, W175R, S1 77N, S178P, G179D, Y179H, K185E, Y187H, H187Y, A197T, K201E, G203I, S 204T, I205M, A206H, W207R, G211D, S215D, F219W, S220W, S220C, C220S, C220L, C220W, Q221M, A229T, N230K, K233E, G235I, I237M, A238H, S247D , F251W, S252L, S252W, S252C, C252S, C252L, C252W, Q253M, N262K, N26 2G, V273*, A280S, S280A, H286Y, Y286H, V305*, A312S, S312A, Y318H, H3 18Y, T319S, S319T, C322S, S322C, T351S, S351T, C354S, S354C, A364P, S366H, N369P, A396P, S398H, S398P, D399M, N401P, S430P, D431M, N438Y , N738S, Y440N, S446P, N452D, T453K, A456S, G458D, P463T, P463L, N464D, Y465N, W468C, N470Y, N470S, Y472N, P476L, S478P, N484D, T485K, A488S, G490D, S490T, P495T, P495L, N496D, Y497N, S499P, W500C, T504N, P508L, S508T, S522T, S531P, T536N, S540T, or any combination thereof, and the variant polypeptide has β-amylase activity.

[0011] A variant polypeptide having β-amylase activity, wherein the variant polypeptide is selected from the group consisting of: (i) the variant polypeptide is at least 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, wherein the variant polypeptide has a specificity for SEQ ID NO: NO: 2 amino acid sequence, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; (b1) D19I, L27C, I28A, S145N, G203I, S220L, A364P, N369P, S398P; (c1) D19I, L27H, Q221M; (d1) D25K, A364P, N369P, S398P; (e1) D25K, A364P, N369P, S398P 9P, S398P; (f1) D25K, G203I, A364P, N369P, S398P; (g1) D25K, I28A, S220L, Q221M, A364P, N369P; (h1) D25K, L27C, G20 3I, A364P, N369P, S398P; (i1) D25K, L27C, G203I, A364P, N369P, S398P; (j1) D25K, L27C, G203I, Q221M, A364P, N369P, S 398P; (k1) D25K, L27C, I28A, A141R, A364P, N369P, S398P; (l1) D25K, L27C, I28A, A141R, A364P, N369P, S398P; (m1) D2 5K, L27C, S145N, S220L, A364P, N369P, S398P; (n1) D25K, L27C, S220L, A364P, N369P, S398P; (o1) D25P, L27H, I28A, Q22 1M; (p1) G203I, A364P, N369P, S398P; (q1) K24E, D25K, L27C, A364P, N369P; (r1) K24E, D25K, L27C, I28A, A141R, A364P, N369P, S398P; (s1) K24E, D25P, L27H, A141R, G203I, S220L, S398P; (t1) K24E, L27H, I28A, S220L, A364P, N369P, S398P;<h2 style=";text-align:left;direction:ltr">(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M ;(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P N438Y, N464D, P476L;(z1)D25K, A364P, N369P, S398P, P463L, Y465N, W468C;(a2)D25K, A364P, N369P, S398P, A456S;(b2)D25K, A364P, N369P, S3 98P, N438Y, Y440N;(c2)D25K, A364P, N369P, S398P, G458D;(d2)D25K, A364P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D 25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h2)D25K, A364P, N369P, S398P, N452D;(i2)D25K, A364P, N369P, S398P N438S;(j2)D25K、A364P、N369P、S398P、T453K;(k2)D25K、A364P、N369P、S398P、S446P;(l2)D25K、A364P、N369P、S398P、W468C;(m2)D25K、L27C S220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N369P, S398P, E48D, N143Y, S146P, K201E;(o2)D25K, L27C, S220L, A3 64P, N369P, S398P, N107S, S172T, W175R;(p2)D25K, L27C, S220L, A364P, N369P, S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398 P, P463T;(r2)D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2)D25K, L27C, S220L, A364P, N369P, S398P, P463L, Y465N, W468C;(t2)D25K, L27C, K153E, K201E, S220L, A364P, N369P, S398P, N438Y, N464D, P476L; (u 2)D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q22 1M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (ii) the variant polypeptide is ID NO:3 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:3, wherein the variant polypeptide has a NO:3 amino acid sequence of the combination of modifications, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; (b1) D19I, L27C, I28A, S145N, G203I, S220L, A364P, N369P, S398P; (c1) D19I, L27H, Q221M; (d1) D25K, A364P, N369P, S398P; (e1) D25K, A364P, N369P, S398P; (f1) D25K, G203I, A364P, N369P, S398P; (g1) D25K, I28A, S220L, Q221M , A364P, N369P; (h1) D25K, L27C, G203I, A364P, N369P, S398P; (i1) D25K, L27C, G203I, A364P, N369P, S398P; (j1) D25K, L27C, G203I, Q221M, A364P, N369P, S398 P; (k1) D25K, L27C, I28A, A141R, A364P, N369P, S398P; (l1) D25K, L27C, I28A, A1 41R, A364P, N369P, S398P; (m1) D25K, L27C, S145N, S220L, A364P, N369P, S398P;<h2 style=";text-align:left;direction:ltr">(n1)D25K、L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N36 9P;(r1)K24E、D25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E 、D25P、L27H、A141R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、 A364P、N369P、S398P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S39 8P;(w1)T101N、D399M;(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K A364P, N369P, S398P, N438Y, N464D, P476L;(z1)D25K, A364P, N369P, S398P, P463L, Y465N, W468C;(a2)D25K, A364P, N369P, S398P, A456S;(b2) D25K, A364P, N369P, S398P, N438Y, Y440N;(c2)D25K, A364P, N369P, S398P, G458D;(d2)D25K, A364P, N369P, S398P, S508T;(e2)D25K, A364P, N36 9P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h2)D25K, A364P, N369P, S398P, N452D;(i2)D 25K, A364P, N369P, S398P, N438S;(j2)D25K, A364P, N369P, S398P, T453K;(k2)D25K, A364P, N369P, S398P, S446P;(l2)D25K, A364P, N369P, S398 P、W468C;(m2)D25K、L27C、S220L、A364P、N369P、S398P、K153E、K201E;(n2)D25K、L27C、S220L、A364P、N369P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K, L27C, S220L, A364P, N369P, S398P, N107S, S172T, W175R; (p2) D25K, L27 C. S220L, A364P, N369P, S398P, Q105L, N119D; (q2) D25K, L27C, S220L, A364P, N369 P, S398P, P463T; (r2) D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476 L; (s2) D25K, L27C, S220L, A364P, N369P, S398P, P463L, Y465N, W468C; (t2) D25K, L2 7C, K153E, K201E, S220L, A364P, N369P, S398P, N438Y, N464D, P476L; (u2) D25K, L2 7C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A36 wherein the variant polypeptide has β-amylase activity; and (iii) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:4. NO:4 amino acid sequence, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; (b1) D19I, L27C, I28A, S145N, G203I, S220L, A364P, N369P, S398P; (c1) D19 I, L27H, Q221M; (d1) D25K, A364P, N369P, S398P; (e1) D25K, A364P, N369P, S398P; (f 1)D25K, G203I, A364P, N369P, S398P; (g1) D25K, I28A, S220L, Q221M, A364P, N369P;<h2 style=";text-align:left;direction:ltr">(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P;(j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1) D25K, L27C, I28A, A141R, A364P, N369P, S398P;(l1)D25K, L27C, I28A, A141R, A364P, N369P, S398P;(m1)D25K, L27C, S145N, S220L, A364P, N369P, S398 P;(n1)D25K、L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q22 1M;(p1)G203I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P; (r1)K24E、D25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、A364P、N3 69P、S398P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101 N、D399M;(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、 S398P, N438Y, N464D, P476L;(z1)D25K, A364P, N369P, S398P, P463L, Y465N, W468C;(a2)D25K, A364P, N369P, S398P, A456S;(b2)D25K, A364P, N369P, S 398P, N438Y, Y440N;(c2)D25K, A364P, N369P, S398P, G458D;(d2)D25K, A364P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h2)D25K, A364P, N369P, S398P, N452D;(i2)D25K, A364P, N369P, S398P, N438S;(j2)D25K, A364P, N369P, S398P, T453K; (k2) D25K, A364P, N369P, S398P, S446P; (l2) D25K, A364P, N369P, S398P, W 468C; (m2) D25K, L27C, S220L, A364P, N369P, S398P, K153E, K201E; (n2) D25K, L27C, S220L, A364P, N369P, S398P, E 48D, N143Y, S146P, K201E; (o2) D25K, L27C, S220L, A364P, N369P, S398P, N107S, S172T, W175R; (p2) D25K, L27C, S2 20L, A364P, N369P, S398P, Q105L, N119D; (q2) D25K, L27C, S220L, A364P, N369P, S398P, P463T; (r2) D25K, L27C, S22 0L, A364P, N369P, S398P, N438Y, N464D, P476L; (s2) D25K, L27C, S220L, A364P, N369P, S398P, P463L, Y465N, W468C ;(t2)D25K, L27C, K153E, K201E, S220L, A364P, N369P, S398P, N438Y, N464D, P476L; (u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (iv) the variant polypeptide is An amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:5, wherein the variant polypeptide has a combination of amino acid modifications to the amino acid sequence of SEQ ID NO:5, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P;<h2 style=";text-align:left;direction:ltr">(b1)D19I、L27C、I28A、S145N、G203I、C220L、A364P、N369P、S398P;(c1)D19 I、L27H、Q221M;(d1)D25K、A364P、N369P、S398P;(e1)D25K、A364P、N369P、S 398P;(f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、C220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L 27C, G203I, A364P, N369P, S398P;(j1)D25K, L27C, G203I, Q221M, A364P, N369P, S398P;(k1)D25K, L27C, I28A, A141R, A364P, N369P, S398P;(l1)D25K L27C、I28A、A141R、A364P、N369P、S398P;(m1)D25K、L27C、S145N、C220L、A3 64P、N369P、S398P;(n1)D25K、L27C、C220L、A364P、N369P、S398P;(o1)D25P、 L27H, I28A, Q221M;(p1)G203I, A364P, N369P, S398P;(q1)K24E, D25K, L27C, A364P, N369P;(r1)K24E, D25K, L27C, I28A, A141R, A364P, N369P, S398P;( s1)K24E、D25P、L27H、A141R、G203I、C220L、S398P;(t1)K24E、L27H、I28A、C 220L、A364P、N369P、S398P;(u1)N143D、A197T;(v1)C220L、A364P、N369P、S 398P;(w1)T101N、D399M;(x1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398 P, P463L, Y465N, W468C;(a2) D25K, A364P, N369P, S398P, A456S;(b2) D25K, A364P, N369P, S398P, N438Y, Y440N;(c2) D25K, A364P, N369P, S398P, G458D;(d2) D25K, A364P, N369P, S398P, S508T; (e2) D25K, A364P, N369P, S398P, T504N; (f2) D25K, A364P, N369P, S398P, S490T; (g2) D25K, A364P, N369P, S398P, S499P; (h2) D25K, A364P, N369P, S398P, N452D; (i2) D25K, A364P, N369P, S398P, N438S; (j2) D25K, A364P, N369P, S398P, T453K; (k2) D25K, A364P, N369P, S398P, S446P; (l2) D25K, A364P, N369P, S398P, W468C; (m2) D25K, L27C, C220L, A364P, N369P, S398P, K153E, K201E; (n2) D25K, L27C, C220L, A364P, N369P, S398P, E48D, N143Y, S146P, K201E; (o2) D25K, L27C, C220L, A364P, N369P, S398P, N107S, S172T, W175R; (p2) D25K, L27C, C220L, A364P, N369P, S398P, Q105L, N119D; (q2) D25K, L27C, C220L, A364P, N369P, S398P, P463T; (r2) D25K, L27C, C220L, A364P, N369P, S398P, N438Y, N464D, P476L; (s2) D25K, L27C, C220L, A364P, N369P, S398P, P463L, Y465N, W468C; (t2) D25K, L27C, K153E, K201E, C220L, A364P, N369P, S398P, N438Y, N464D, P476L; (u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β - amylase activity;(vi) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:6, wherein the variant polypeptide has a sequence identical to that of SEQ ID NO:6. NO:6 amino acid sequence, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; (b1) D19I, L27C, I28A, S145N, G203I, S220L, A364P, N369P, S398P; (c1) D19I, L27H, Q221M; (d1) D25K, A364P, N369P, S398P; (e1) D25K, A364P, N369P, S 398P; (f1) D25K, G203I, A364P, N369P, S398P; (g1) D25K, I28A, S220L, Q221M, A364P, N369P; (h1) D25K, L27C, G203I, A36 4P, N369P, S398P; (i1) D25K, L27C, G203I, A364P, N369P, S398P; (j1) D25K, L27C, G203I, Q221M, A364P, N369P, S398P; (k1) )D25K, L27C, I28A, A141R, A364P, N369P, S398P; (l1) D25K, L27C, I28A, A141R, A364P, N369P, S398P; (m1) D25K, L27C, S1 45N, S220L, A364P, N369P, S398P; (n1) D25K, L27C, S220L, A364P, N369P, S398P; (o1) D25P, L27H, I28A, Q221M; (p1) G203I , A364P, N369P, S398P; (q1) K24E, D25K, L27C, A364P, N369P; (r1) K24E, D25K, L27C, I28A, A141R, A364P, N369P, S398P; (s 1)K24E, D25P, L27H, A141R, G203I, S220L, S398P; (t1) K24E, L27H, I28A, S220L, A364P, N369P, S398P; (u1) N143D, A197T;<h2 style=";text-align:left;direction:ltr">(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M;(X1)D25K、A364P 、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P 476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W468C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y4 40N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364P、N369P、S398P、S508T;(e2)D25K、A364P、N369P、S398P、T504N;(f2)D25K、A364P、 N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h2)D25K, A364P, N369P, S398P, N452D;(i2)D25K, A364P, N369P, S398P, N438S; j2)D25K, A364P, N369P, S398P, T453K;(k2)D25K, A364P, N369P, S398P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, S220L A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N369P, S398P, E48D, N143Y, S146P, K201E;(o2)D25K, L27C, S220L, A364P N369P, S398P, N107S, S172T, W175R;(p2)D25K, L27C, S220L, A364P, N369P, S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398P P463T;(r2)D25K、L27C、S220L、A364P、N369P、S398P、N438Y、N464D、P476L;(s2)D25K、L27C、S220L、A364P、N369P、S398P、P463L、Y465N、W468C;(t2)D25K, L27C, K153E, K201E, S220L, A364P, N369P, S398P, N438Y, N464D, P476L; (u2 )D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221 M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (vii) the variant polypeptide is ID NO:7 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:7, wherein the variant polypeptide has a NO:7 amino acid sequence of the combination of amino acid modifications, and the combination is selected from the group consisting of: (a1) D51I, L59C, A173R, A396P, N401P, S430P; (b1) D51I, L59C, I60A, S177N, G235I, S252L, A396P, N401P, S430P; (c1) D51I, L59H, Q253M; (d1) D57K, A396P, N401P, S430P; (e1) D57K, A396P, N401P, S430P; (f1) D57K, G235I, A396P, N401P, S430P; (g1) D57K, I60A, S252L, Q253M , A396P, N401P; (h1) D57K, L59C, G235I, A396P, N401P, S430P; (i1) D57K, L59C, G235I, A396P, N401P, S430P; (j1) D57K, L59C, G235I, Q253M, A396P, N401P, S430 P; (k1) D57K, L59C, I60A, A173R, A396P, N401P, S430P; (l1) D57K, L59C, I60A, A1 73R, A396P, N401P, S430P; (m1) D57K, L59C, S177N, S252L, A396P, N401P, S430P;<h2 style=";text-align:left;direction:ltr">(n1)D57K、L59C、S252L、A396P、N401P、S430P;(o1)D57P、L59H、I60A、Q253M;(p1)G235I、A396P、N401P、S430P;(q1)K56E、D57K、L59C、A396P、N3 69P;(r1)K56E、D57K、L59C、I60A、A173R、A396P、N401P、S430P;(s1)K56 E、D57P、L59H、A173R、G235I、S252L、S430P;(t)K56E、L59H、I60A、S252L、 A396P, N401P, S430P;(u1)N175D, A229T;(v1)S252L, A396P, N401P, S430P;(w1)T133N, D431M;(x1)D57K, A396P, N401P, S430P, P495T;(y1)D57K A396P, N401P, S430P, N470Y, N496D, P508L;(z1)D57K, A396P, N401P, S430P, P495L, Y497N, W500C;(a2)D57K, A396P, N401P, S430P, A488S;(b2) D57K, A396P, N401P, S430P, N470Y, Y472N;(c2)D57K, A396P, N401P, S430P, G490D;(d2)D57K, A396P, N401P, S430P, S540T;(e2)D57K, A396P, N40 1P, S430P, T540N;(f2)D57K, A396P, N401P, S430P, S522T;(g2)D57K, A396P, N401P, S430P, S531P;(h2)D57K, A396P, N401P, S430P, N484D;(i2)D 57K, A396P, N401P, S430P, N470S;(j2)D57K, A396P, N401P, S430P, T485K;(k2)D57K, A396P, N401P, S430P, S478P;(l2)D57K, A396P, N401P, S430 P、W500C;(m2)D57K、L59C、S252L、A396P、N401P、S430P、K185E、K233E;(n2)D57K、L59C、S252L、A396P、N401P、S430P、E48D、N175Y、S178P、K233E;(o2)D57K, L59C, S252L, A396P, N401P, S430P, N139S, S204T, W207R; (p2) D57K, L59C, S252L, A396P, N401P, S430P, Q137L, N151D; (q2) D57K, L59C, S252L, A396 P, N401P, S430P, P495T; (r2) D57K, L59C, S252L, A396P, N401P, S430P, N470Y, N 496D, P508L; (s2)D57K, L59C, S252L, A396P, N401P, S430P, P495L, Y497N, W500C ;(t2)D57K, L59C, K185E, K233E, S252L, A396P, N401P, S430P, N470Y, N496D, P5 08L; (u2)D57K, L59C, G235I, Q253M, A396P, N401P, S430P, P495T; (v2) D57K, L5 9C, G235I, Q253M, A396P, N401P, S430P, N470Y, N496D, P508L; (w2) D57K, L59C, G235I, Q253M, A396P, N401P, S430P, P495L, Y497N, W500C; wherein the variant polypeptide has β-amylase activity. ;

[0012] A variant polypeptide having β-amylase activity, wherein the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, and the variant polypeptide has increased enzyme activity, thermal stability, pH stability or any combination thereof when compared to the β-amylase of SEQ ID NO:2.

[0013] The variant polypeptide, wherein the variant polypeptide is a fragment of the full-length amino acid sequence, and the fragment has β-amylase activity.

[0014] A variant polypeptide comprising a hybrid of at least one variant polypeptide and a second polypeptide having amylase activity, wherein the hybrid has beta-amylase activity.

[0015] A composition comprising a variant polypeptide.

[0016] A composition comprising a variant polypeptide and at least a second enzyme.

[0017] A composition comprising a variant polypeptide and at least a second enzyme, wherein the second enzyme is selected from the group consisting of a second beta-amylase, a lipase, an alpha-amylase, a G4-amylase, a xylanase, a protease, a cellulase, a glucoamylase, an oxidoreductase, a phospholipase, and a cyclodextrin glucanotransferase.

[0018] A method for producing the variant polypeptide comprises: providing a template nucleic acid sequence encoding the variant polypeptide, transforming the template nucleic acid sequence into an expression host, culturing the expression host to produce the variant polypeptide, and purifying the variant polypeptide.

[0019] A method for producing the variant polypeptide, wherein the template nucleic acid is a variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide encodes a polypeptide having β-amylase activity.

[0020] A method for producing the variant polypeptide, wherein the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system and a synthetic expression system.

[0021] A method for producing the variant polypeptide, wherein the bacterial expression system is selected from Escherichia coli (E. coli), Bacillus, Pseudomonas and Streptomyces.

[0022] A method for producing the variant polypeptide, wherein the yeast expression system is selected from Candida, Pichia, Saccharomyces, and Schizosaccharomyces.

[0023] A method for producing the variant polypeptide, wherein the fungal expression system is selected from Penicillium, Aspergillus, Fusarium, Myceliopthora, Rhizomucor, Rhizopus, Thermomyces and Trichoderma.

[0024] A method for preparing dough or a baked product prepared from the dough, the method comprising adding one of the variant polypeptides to the dough and baking it. A method for preparing dough or a baked product, comprising: providing the variant polypeptide, providing dough, contacting the variant polypeptide with the dough, and optionally baking the dough to produce a baked product.

[0025] A method for using a variant polypeptide for treating starch. A method for treating starch, comprising: providing a variant polypeptide, providing starch, and contacting the variant polypeptide with the starch, wherein the variant polypeptide hydrolyzes the starch.

[0026] A method of using a variant polypeptide for cleaning or laundering fabrics, hard surfaces, or dishes. A method for cleaning or laundering fabrics, hard surfaces, or dishes comprising: providing a variant polypeptide, providing a fabric, hard surface, or dish, contacting the variant polypeptide with the fabric, hard surface, or dish, wherein the variant polypeptide cleans or launders the fabric, hard surface, or dish.

[0027] A method for using a variant polypeptide for producing ethanol. A method for producing ethanol, comprising: providing a variant polypeptide, providing a raw material, and contacting the variant polypeptide with the raw material, wherein the variant polypeptide hydrolyzes the raw material into sugars, thereby inducing the sugars to produce ethanol.

[0028] A method of using a variant polypeptide for treating an oil well. A method of treating an oil well, comprising: providing a variant polypeptide, providing an oil well fracturing fluid; contacting the variant polypeptide with the oil well fracturing fluid, wherein the variant polypeptide hydrolyzes the oil well fracturing fluid.

[0029] A method of using the variant polypeptide according to any one of claims 1 to 7 for treating pulp or paper comprising: providing a variant polypeptide, providing pulp or paper, contacting the variant polypeptide with the pulp or paper, wherein the variant polypeptide hydrolyzes the pulp or paper.

[0030] A method of using a variant polypeptide for feeding an animal. A method of making an animal feed, comprising: providing a variant polypeptide, providing an animal feed, and contacting the variant polypeptide with the animal feed, wherein the variant polypeptide is a part of the animal feed.

[0031] A method for using a variant polypeptide for making syrup. A method for making syrup, comprising: providing a variant polypeptide, providing a sugar, and contacting the variant polypeptide with the sugar, wherein the variant polypeptide hydrolyzes the sugar to produce syrup. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The pH profiles of the variant β-amylases are shown.

[0033] Figure 2The results of bread firmness and elasticity for the 20-day test are shown. DETAILED DESCRIPTION

[0034] An enzyme is a biomolecule comprising an amino acid sequence, wherein the enzyme catalyzes a reaction. Enzyme names are known to those skilled in the art based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzyme names include: EC (Enzyme Commission) number, suggested name, alternative name (if any), catalytic activity, and other factors. Enzymes are also referred to as polypeptides, proteins, peptides, amino acid sequences, or are identified by SEQ ID NO. Alternative names for enzymes may be used interchangeably.

[0035] Different classes of enzymes are known to be suitable for use in baking, including: α-amylases (EC 3.2.1.1); β-amylases (3.2.1.2), glucan 1,4-α-maltotetraosylase (EC 3.2.1.60), also known as exomaltotetraosylase, G4-amylase; glucan 1,4-α-maltohydrolase (EC 3.2.1.133), also known as maltogenic α-amylase; endo-1,4-β-xylanase (EC 3.2.1.8); oxidoreductases; phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); galactolipase (EC 3.1.1.26), cellulase (EC 3.2.1.4), transglutaminase (EC 3.2.1.60), glutaminase (EC 3.2.1.61), galactolipase (EC 3.2.1.62), glutaminase (EC 3.2.1.63), glutaminase (EC 3.2.1.64), galactolipase (EC 3.2.1.65), glutaminase (EC 3.2.1.66), glutaminase (EC 3.2.1.67), glutaminase (EC 3.2.1.68), glutaminase (EC 3.2.1.69), glutaminase (EC 3.2.1.70), glutaminase (EC 3.2.1.71), glutaminase (EC 3.2.1.72), glutaminase (EC 2.3.2.13), phytases (EC 3.1.3.8; 3.1.3.26; and 3.1.1.72) and proteases. Enzymes are used as food ingredients, food additives and / or processing aids.

[0036] β-amylase (EC 3.2.1.2) hydrolyzes preformed (1->4)-α-D-glycosidic bonds in polysaccharides to remove consecutive maltose units from the non-reducing end of the chain. It acts on starch, glycogen and related polysaccharides and oligosaccharides that produce β-maltose by reversal. This enzyme is widely used in the fermentation industry for making caramel, maltose, maltodextrin and brewing beer, alcohol and vinegar. β-amylases are characterized by higher plant and microbial sources, for example, β-amylases from microbial sources are: Bacillus acidopullulyticus (U.S. Pat. No. 4,970,158), Bacillus flexus (Matsunaga, Okada and Yamagat, H. and Tsukagishi, N. 1987, Journal of Bacteriology (J. Bacteriol.) (169) 1564-1570) and U.S. Pat. No. 8,486,682. Other β-amylases from microbial sources are: Clostridium thermosulfurogenes (Kitaoto, 1988, Kitamoto, N., Yamagata, H., Kato, T., Tsukagoshi, N. and Udaka, S. 1998. Journal of Bacteriology (170) 5848-5854), U.S. Patent Application Publication 2012 / 0225164, WO2015 / 021600, WO2015 / 021601, EP0337090A1 and Thermoanaerobacterium thermosulfurigenes JP01218589. Based on the β-amylase from Clostridium thermosulfurogenes, an acid-resistant β-amylase was developed (CN103695386 and CN 103881993).

[0037] Additionally, amylases are disclosed in patents and published patent applications including WO2002 / 068589, WO2002 / 068597, WO2003 / 083054, WO2004 / 042006, WO2008 / 080093, WO2013 / 116175, and WO2017 / 106633.

[0038] Commercially available amylases for food processing and baking include: Available from DSM and Available from DuPont Max-LIFE TM , and and commercially available from Novozymes and

[0039] Lipases (EC 3.1.1.3) are hydrolases known to cleave ester bonds in lipids. Lipases include phospholipases, triacylglycerol lipases, and galactolipases. Lipases have been identified from plants, mammals, and microorganisms, including, but not limited to, Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, Cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum, Fusarium culmorum lipases.

[0040] In addition, various lipases, phospholipases and galactolipases have been disclosed in patents and published patent applications, including but not limited to: WO1993 / 000924, WO2003 / 035878, WO2003 / 089620, WO2005 / 032496, WO2005 / 086900, WO2006 / 031699, WO2008 / 036863 and WO2011 / 046812.

[0041] Commercially available lipases for food processing and baking include, but are not limited to: LIPOPAN TM , NOOPAZYME, LIPOPANMAX, LIPOPAN Xtra (available from Novozymes); PANAMORE, CAKEZYME and BAKEZYME (available from DSM); and GRINDAMYL EXEL 16, GRINDAMYL POWERBAKE and TS-E 861 (available from DuPont / Danisco).

[0042] The "parent" polypeptide amino acid sequence is a starting sequence for introducing a mutation into the sequence (e.g., by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) to produce a "variant" of the parent polypeptide amino acid sequence. Parents include: wild-type polypeptide amino acid sequences or synthetically produced polypeptide amino acid sequences, which are used as starting sequences for introducing (other) changes.

[0043] A "variant polypeptide" refers to an enzyme that differs from its parent in its amino acid sequence. Variant polypeptides are described using the nomenclature and abbreviations of single amino acid molecules in accordance with the IUPAC recommendations for single-letter or three-letter amino acid abbreviations.

[0044] "Substitution" is described by providing the original amino acid and then the position number within the amino acid sequence. For example, the substitution of amino acid residue 24 means that the amino acid of the parent at position 24 can be replaced by any of 19 other amino acid residues. In addition, substitutions can be described by providing the original amino acid, then the position number within the amino acid sequence, and then the specific substituted amino acid. For example, the replacement of histidine at position 120 with alanine is called "His120Ala" or "H120A". Combinations of substitutions are described by inserting commas between amino acid residues, for example: K24E, D25P, L27H, A141R, G203I, S220L, S398P; when compared to the parent polypeptide, it represents a combination of seven different amino acid residue substitutions. Variants with substitutions in the case of amino acid changes can also be applied to nucleic acid modifications, such as by substitution.

[0045] Variant polypeptides with amino acid substitutions, wherein the substitutions may be conservative amino acid substitutions. "Conservative amino acid substitutions" or "related amino acids" means replacing an amino acid residue in an amino acid sequence with a different amino acid residue having similar properties at the same position compared to the parent amino acid sequence. Some examples of conservative amino acid substitutions include, but are not limited to, replacing positively charged amino acid residues with different positively charged amino acid residues; replacing polar amino acid residues with different polar amino acid residues; replacing non-polar amino acid residues with different non-polar amino acid residues, replacing basic amino acid residues with different basic amino acid residues, or replacing aromatic amino acid residues with different aromatic amino acid residues. A list of related amino acids is given in the table below (see, e.g., Creighton (1984) Proteins. WH Freeman and Company (Eds)).

[0046] Examples of conservative amino acid substitutions:

[0047] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

[0048] WIPO standard ST.25 (1998) provides that amino acid residues should be represented in the sequence table using the following three-letter symbols, with the first letter capitalized. The following table provides an overview of amino acid identifiers and the corresponding DNA codons encoding amino acids using standard genetic standards. The DNA codons encoding amino acid residues may vary depending on the organism used, and slightly different genetic code translation tables are applied. NCBI maintains a compilation of such non-standard code translation tables. For reference, see, for example, https: / / www.ncbi.nlm.nih.gov / Taxonomy / Utils / wprintgc.cgi.

[0049]

[0050]

[0051] Variant polypeptides having β-amylase activity may be "mature polypeptides". Mature polypeptides refer to enzymes in their final form, including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modification, C-terminal modification, signal sequence deletion. Mature polypeptides may vary depending on the expression system, vector, promoter and / or production method.

[0052] "Enzyme activity" means at least one catalytic action performed by an enzyme. Enzyme activity is expressed as the number of units per milligram of enzyme (specific activity), or as the number of molecules of substrate converted per molecule of enzyme per minute (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, such as proteases that exert proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases that exert lipolytic activity by hydrolytic cleavage of ester bonds, etc.

[0053] During storage or operational use of the enzyme, the enzyme activity changes. The term "enzyme stability" relates to the retention of enzyme activity over time during storage or operation.

[0054] In order to determine and quantify the change in the catalytic activity of an enzyme stored or used over time under certain conditions, the "initial enzyme activity" is measured at time 0 (100%) and at a later time point (x%) under defined conditions. By comparing the measured values, the extent of potential loss of enzyme activity can be determined. The extent of enzyme activity loss determines the stability or instability of the enzyme.

[0055] Parameters which influence the enzymatic activity and / or the storage stability and / or the processing stability of the enzyme are, for example, pH, temperature and the presence of oxidizing substances:

[0056] Variant polypeptides may have activity at any single point within a wide pH range, from about pH 4.0 to about pH 12.0. Variant polypeptides having beta-amylase activity have activity within the range of pH 4.0 to pH 11.0, pH 4.0 to pH 10.0, pH 4.0 to pH 9.0, pH 4.0 to pH 8.0, pH 4.0 to pH 7.0, pH 4.0 to pH 6.0, or pH 4.0 to pH 5.0. The variant polypeptides having β-amylase activity have β-amylase activity at pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH9.0, pH 9.1, pH 9.2, pH 9.3, pH 9.4, pH 9.5, pH 9.6, pH9.7, pH 9.8, pH 9.9, pH 10.0, pH10.1, pH 10.2, pH 10.3, pH 10.4, pH 10.5, pH 10.6, pH 10.7, pH 10.8, pH 10.9, pH 11.0, pH 11.1, pH 11.2, pH 11.3, pH 11.4, pH 11.5, pH 11.6, pH 11.7, pH The invention relates to a novel molecule of the present invention and is active at pH 11.8, pH 11.9, pH 12.0, pH 12.1, pH 12.2, pH 12.3, pH 12.4 and pH 12.5, pH 12.6, pH 12.7, pH 12.8, pH 12.9 and higher pH.

[0057] "pH stability" refers to the ability of a protein to function within a specific pH range. Typically, most enzymes function under conditions of higher or lower pH ranges.

[0058] Variant polypeptides may be active over a wide range of temperatures used at any time during the baking process, wherein the temperature is any point in the range of about 20° C. to about 60° C. Variant polypeptides having beta-amylase activity are active in a temperature range of 20° C. to 55° C., 20° C. to 50° C., 20° C. to 45° C., 20° C. to 40° C., 20° C. to 35° C., 20° C. to 30° C., or 20° C. to 25° C. Variant polypeptides having beta-amylase activity have activity at a temperature of at least 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C or more.

[0059] "Thermal stability" or "thermostability" refers to the ability of a protein to function within a certain temperature range. In general, most enzymes have a limited temperature range over which they function. In addition to enzymes that function at a moderate range of temperatures (e.g., room temperature), there are enzymes that can function at very high or very low temperatures. Thermostability is characterized by a temperature range called T 50 The value (also called half-life, see above) of 50 Indicates the temperature at which 50% of the activity remains after thermal inactivation for a certain period of time compared to a reference sample that has not undergone thermal treatment.

[0060] "Thermal tolerance" or "thermotolerance" refers to the ability of a protein to function after being exposed to a specific temperature (e.g., very high or very low temperatures). A thermotolerant protein may not function at the exposure temperature, but will function once it returns to a favorable temperature.

[0061] "Purified" means that the material is in a relatively pure state, such as at least about 90% pure, at least about 95% pure, or at least about 98% or 99% pure. Preferably, "purified" means that the material is in a 100% pure state.

[0062] "Synthetic" or "artificial" compounds are produced by in vitro chemical or enzymatic synthesis. Variant nucleic acids can be produced using optimal codon usage for a host organism (e.g., a yeast cell host or other selected expression host, or a variant protein sequence having amino acid modifications (e.g., substitutions) compared to a parent protein sequence), e.g., to optimize the properties of a polypeptide.

[0063] The term "non-naturally occurring" refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism or other material that is not found in its original environment or source, although it may have been originally derived from its original environment or source and then derived from a non-naturally occurring process reproduced under laboratory conditions.

[0064] "Sequence identity", "sequence identity %", "identity %" or "sequence alignment" means the comparison of a first amino acid sequence to a second amino acid sequence or a first nucleic acid sequence to a second nucleic acid sequence, and is calculated as a percentage based on the comparison. This calculation result can be described as "identity percentage" or "ID percentage".

[0065] Typically, sequence alignment can be used to calculate sequence identity by one of two different approaches. In the first approach, in the final sequence identity calculation, both the mismatch of a single position and the gap of a single position are counted as inconsistent positions. In the second approach, in the final sequence identity calculation, the mismatch of a single position is counted as an inconsistent position; however, in the final sequence identity calculation, the gap of a single position is not counted as (ignored) inconsistent positions. In other words, in the second approach, the gap is ignored in the final sequence identity calculation. The difference between these two approaches (counting the gap as an inconsistent position vs. ignoring the gap at a single position) causes the sequence identity value between the two sequences to change.

[0066] Sequence identity is determined by a program that generates an alignment and counts mismatches at a single position and gaps at a single position as inconsistent positions in the final sequence identity calculation, calculating the identity. For example, the program Needle (EMBOS), which has implemented the algorithm of Needleman and Wunsch (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), calculates sequence identity by first generating an alignment between a first sequence and a second sequence, then counting the number of consistent positions over the alignment length, then dividing the number of consistent residues by the alignment length, and then multiplying this number by 100 to generate a sequence identity % [sequence identity % = (number of consistent residues / alignment length) × 100)].

[0067] Sequence identity can be calculated from pairwise alignments showing the two sequences over their full length, thus showing the first sequence and the second sequence over their full length (" Global sequence consistency ”). For example, the program Needle (EMBOSS) generates such an alignment; % sequence identity = (number of identical residues / alignment length) × 100)].

[0068] Sequence identity ("sequence identity") can be calculated from pairwise alignments showing only local regions of the first or second sequence. Bureau Departmental consistency ”). For example, the program Blast (NCBI) generates such an alignment; % sequence identity = (number of identical residues / alignment length) × 100)].

[0069] The sequence alignment is calculated, and mismatches at a single position are counted as inconsistent positions in the final sequence consistency calculation; however, gaps at a single position are not counted (ignored) as inconsistent positions in the final sequence consistency calculation. The sequence alignment is generated by using the algorithm of Needleman and Wunsch (Journal of Molecular Biology (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite; EMBOSS) is used with the program default parameters (gap opening = 10.0, gap extension = 0.5 and matrix = EBLOSUM62). The sequence identity can then be calculated from the alignment of the two sequences displayed over their full length, thus displaying the first sequence and the second sequence in their full length (" Global sequence consistency ”). For example: % identity = (number of identical residues / alignment length) × 100)].

[0070] Variant polypeptides are described as having an amino acid sequence at least n% identical to the amino acid sequence of the respective parent enzyme, wherein "n" is an integer between 10 and 100. Variant polypeptides include enzymes that are at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 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% identical when compared to the full-length amino acid sequence of the parent enzyme, wherein the enzyme variant has enzymatic activity. As used herein, a "fragment" or "subsequence" is a portion of a polynucleotide or amino acid sequence. A fragment or subsequence may retain or encode at least one functional activity of the sequence to which it is associated.

[0071] "Functional fragment" refers to any nucleic acid or amino acid sequence that contains only a portion of the full-length nucleic acid or full-length amino acid sequence, respectively, but still has the same or similar activity and / or function. The fragment accounts for at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the original sequence. Compared with the original nucleic acid or original amino acid sequence, the functional fragment contains continuous nucleic acids or amino acids, respectively.

[0072] The variant polypeptide having β-amylase activity may be a hybrid of more than one β-amylase. "Hybrid" or "chimeric" or "fusion protein" means that the domain of the first variant polypeptide β-amylase is combined with the domain of the second β-amylase to form a hybrid amylase, and the hybrid has amylase activity. The domain of the variant polypeptide having β-amylase activity can be combined with the domain of a commercially available amylase, such as from AB Enzymes. Available from DSM and Available from DuPont Max-LIFE TM , and and commercially available from Novozymes and Additionally, domains from various amylases can be recombined into a single enzyme, wherein the enzyme has amylase activity.

[0073] Industrial enzymes are typically recombinant proteins produced using bacterial, fungal or yeast expression systems."Expression system" also refers to a host microorganism, an expression host, a host cell, a production organism or a production strain, and each of these terms can be used interchangeably. Examples of expression systems include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably Bacillus subtilis or Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophila (C1), Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei, and yeast, preferably Saccharomyces cerevisiae. cerevisiae). Variant polypeptides having β-amylase activity were produced using the expression systems listed above.

[0074] The term "heterologous" (or exogenous or foreign or recombinant) polypeptide is: (a) a polypeptide that is not native to the host cell. The protein sequence of such a heterologous polypeptide is a synthetic, non-naturally occurring "artificial" protein sequence; (b) a polypeptide that is native to the host cell in which structural modifications, such as deletions, substitutions and / or insertions have been made to alter the native polypeptide; or (c) a polypeptide that is native to the host cell, whose expression has been quantitatively altered, or whose expression is directed by a genomic location that differs from the native host cell due to manipulation of the host cell's DNA by recombinant DNA technology (e.g., a stronger promoter).

[0075] The term "heterologous" (or exogenous or foreign or recombinant) polynucleotide refers to: (a) a polynucleotide that is not native to the host cell; (b) a polynucleotide that is not native to the host cell in which structural modifications have been made, such as deletions, substitutions and / or insertions, to alter the native polynucleotide; (c) a polynucleotide that is native to the host cell, the expression of which is quantitatively altered due to manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or (d) a polynucleotide that is native to the host cell but has not been integrated into its natural genetic environment due to genetic manipulation by recombinant DNA techniques.

[0076] With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally occur in a specific combination with each other.

[0077] "Vector" means any kind of construct suitable for carrying an exogenous polynucleotide sequence to transfer to another cell or to stably or transiently express in a given cell. The term "vector" encompasses any kind of cloning vector, such as but not limited to plasmids, phagemids, viral vectors (e.g., phage), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes or any other vectors specific to a particular host. Low or high copy number vectors are also included. Exogenous polynucleotide sequences typically include coding sequences, which may be referred to as "related genes". Related genes may include introns and exons, depending on the origin of the host cell or the type of target.

[0078] A variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide encodes an enzyme variant polypeptide having β-amylase activity.

[0079] A method for producing a variant polypeptide having β-amylase activity, comprising: providing a template nucleic acid sequence, wherein the template nucleic acid is a variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide encodes a polypeptide having β-amylase activity.

[0080] Transform the template nucleic acid sequence into the expression host

[0081] The expression host is cultivated to produce the variant polypeptide, and the variant polypeptide is purified.

[0082] The template nucleic acid is a variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide is a nucleic acid sequence at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide encodes a polypeptide having β-amylase activity.

[0083] The polypeptide variants having β-amylase activity can be used or formulated alone or in the form of a mixture of enzymes.

[0084] The formulation may be in solid form, e.g., powder, lyophilized preparation, granules, tablet, bar, crystal, capsule, pill, pellet; or in liquid form, e.g., in the form of an aqueous solution, an aerosol, a gel, a paste, a slurry, an aqueous / oil emulsion, a cream, a capsule, or in the form of a vesicular or micellar suspension.

[0085] The variant polypeptide with beta-amylase activity can be used in combination with at least one other enzyme. The other enzymes can be from the same class of enzymes, such as a composition comprising a first beta-amylase and a second beta-amylase. The other enzymes can also be from different classes of enzymes, such as a composition comprising a variant polypeptide with beta-amylase activity and a lipase. The combination with at least one other enzyme can be a composition comprising at least three enzymes. These three enzymes can have enzymes from the same class of enzymes (e.g., a first amylase, a second amylase, and a third amylase); or the enzymes can be from different classes of enzymes, such as a variant polypeptide with beta-amylase activity, a lipase, and a xylanase.

[0086] The second enzyme comprises: α-amylase; β-amylase, glucan 1,4-α-maltotetraose hydrolase, also known as exomaltotetraose hydrolase, G4-amylase; glucan 1,4-α-maltohydrolase, also known as maltogenic α-amylase, cyclodextrin glucanotransferase, glucoamylase; endo-1,4-β-xylanase; xylanase, cellulase, oxidoreductase; phospholipase A1; phospholipase A2; phospholipase C; phospholipase D; galactolipase, triacylglycerol lipase, arabinofuranosidase, transglutaminase, pectinase, pectate lyase, protease or any combination thereof. The enzyme combination is a variant polypeptide with β-amylase activity and a lipase, or the enzyme combination is β-amylase, lipase and xylanase.

[0087] The variant polypeptide having β-amylase activity may be in a composition. The composition comprises a variant polypeptide having β-amylase activity.

[0088] The composition comprises a variant polypeptide having β-amylase activity and a second enzyme.

[0089] Preferably, the second enzyme is selected from the group consisting of a second beta-amylase, a lipase, an alpha-amylase, a G4-amylase, a xylanase, a protease, a cellulase, a glucoamylase, an oxidoreductase, a phospholipase and a cyclodextrin glucanotransferase.

[0090] The composition can be used to prepare baked products.

[0091] The variant polypeptides having β-amylase activity can be used in a method of preparing a dough or a baked product prepared from the dough, the method comprising adding one of the variant enzymes to the dough, and baking it.

[0092] Bread includes, but is not limited to, rolls, buns, pastries, cakes, flatbreads, pizza, bagels, wafers, naan, lavash, pita, focaccia, sourdough, noodles, cookies, fried (donut) tortillas, pancakes, crepes, croutons, and biscuits. Bread can also be in an edible container, such as a cup or cone. Baking bread generally involves mixing ingredients to form dough, kneading, fermentation, shaping, baking, cooling, and storage. The ingredients used to make the dough generally include flour, water, salt, yeast, and other food additives.

[0093] Flour is typically made from wheat and can be milled for different purposes, such as making bread, pastries, cakes, cookies, pasta, and noodles. Alternatives to wheat flour include, but are not limited to, almond flour, coconut flour, chia flour, corn flour, barley flour, spelt flour, soy flour, hemp flour, potato flour, quinoa, teff flour, rye flour, amaranth flour, arrowroot flour, chickpea (garbanzo) flour, cashew flour, flax flour, macadamia flour, millet flour, sorghum flour, rice flour, tapioca flour, and any combination thereof. Flour types are known to vary from region to region and country to country throughout the world.

[0094] Processing flour or dough can include adding inorganic substances, organic substances, such as fatty acids, carbohydrates, amino acids, proteins and nuts. Flour or dough can be pre-treated by cooling, heating, irradiation, agglomeration or freeze drying before baking. In addition, flour or dough can be pre-treated by adding enzymes or microorganisms (such as yeast) before baking.

[0095] Yeast breaks down sugars into carbon dioxide and water. A variety of baking yeasts, generally derived from Saccharomyces cerevisiae, are known to those skilled in the art, including, but not limited to, cream yeast, compressed yeast, cake yeast, active dry yeast, instant yeast, hyperosmotic yeast, rapid yeast, deactivated yeast. Other types of yeast include nutritional yeast, beer yeast, distilling yeast, and wine yeast.

[0096] Sweeteners include, but are not limited to, liquid sugar, syrups, white (granulated) sugar, brown (raw) sugar, honey, fructose, dextrose, glucose, high fructose corn syrup, molasses, and artificial sweeteners.

[0097] Emulsifiers include, but are not limited to, diacetyl tartaric acid esters of monoglycerides (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), ethoxylated mono- and diglycerides (EMG), polysorbates (PS), and succinylated monoglycerides (SMG).

[0098] Other food additives that may be used with baking methods include: lipids, oils, butter, margarine, shortening, milk fat, glycerin, eggs, dairy products, non-dairy substitutes, thickeners, preservatives, colorants, and enzymes.

[0099] During the baking process, the ingredients or additives used for baking can be added separately. The ingredients or additives can also be combined with more than one ingredient or additive to form a premix, and then the premix is ​​added during the baking process. The flour or dough mixture can be prepared before baking, including ready oven dough, packaged dough, or packaged batter.

[0100] Bread products can be modified to meet special dietary requirements, such as sugar-free, gluten-free, low-fat or any combination thereof. The enzymes can extend the shelf life of dough-based products or provide antimicrobial (mold-free) effects.

[0101] Variant polypeptides with β-amylase activity may be suitable for other industrial applications. Variant polypeptides with β-amylase activity are used in detergents, personal care products, in fabric treatment, in pulp and paper treatment, in the production of ethanol, lignocellulosic ethanol or syrups; and as viscosity reducers in the oil field and mining industry.

[0102] "Bakery products" include, for example, bread, crisp rolls, sandwich bread, buns, baguettes, ciabatta, croissant, noodles, and fine bakery products such as donuts, brioche, stollen, cakes, muffins, etc.

[0103] "Dough" is defined as a mixture of flour, salt, yeast and water that may be kneaded, molded, shaped or rolled before baking. In addition, other ingredients such as sugar, margarine, eggs, milk, etc. may also be used. The term includes doughs used to prepare baked goods such as bread, rolls, sandwich bread, baguettes, ciabatta, croissants, sweet yeast doughs, etc.

[0104] "Bread volume" is the volume of a baked product determined by measuring volume and specific volume using a laser scanner (e.g., Micro Stable System's VolscanProfiler). The term also includes volumes determined by measuring the length, width, and height of certain baked products.

[0105] The description in range format is merely for convenience and brevity and should not be construed as a fixed limitation on the scope of the present disclosure. Thus, the description of a range is deemed to have specifically disclosed all possible subranges within the range as well as individual numerical values. For example, a description of a range such as 1 to 6 is deemed to have specifically disclosed subranges within the range (e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) as well as individual numbers, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0106] Other objects, advantages and features of the present disclosure will become apparent from the following description taken in conjunction with the accompanying drawings.

[0107] In the following description, numerous specific details are set forth to provide a thorough description of the present disclosure. However, it will be apparent to those skilled in the art that the methods of the present disclosure may be practiced without one or more of these specific details. In other cases, well-known features and procedures well known to those skilled in the art are not described to avoid obscuring the present disclosure.

[0108] Example 1: PAHBAH analysis

[0109] Quantification of starch hydrolysis by β-amylase and variant enzymes was measured using the 4-hydroxybenzohydrazide method as described in Lever M. (1972) "A new reaction for colorimetric determination of carbohydrates." Anal. Biochem. 47, 273-279 with the following modifications. 112uL of 1% potato amylopectin was reacted with 12.5uL of diluted enzyme at 65°C and samples were taken at 10 minutes. The reaction was then quenched by mixing into 100ul of 1% PAHBAH reagent. The reaction was heated to 95°C for 6 minutes, cooled to room temperature, and the solution absorbance was read at 410nm in a BioTek plate reader.

[0110] Example 2: Residual Activity

[0111] Residual activity was calculated by comparing the activity of each enzyme as measured using the PAHBAH assay before and after heat shock at 82° C. or 88° C. After heating the samples for 10 minutes at 82° C. or 88° C., the samples were cooled to room temperature before testing at 65° C. using the PAHBAH assay.

[0112] Example 3: ThermoFluor Analysis

[0113] The melting point (Tm) of each β-amylase variant was measured in a high throughput manner using the ThermoFluor assay as described in Lo, MC; Aulabaugh, A; Jin, G; Cowling, R; Bard, J; Malamas, M; Ellestad, G (September 1, 2004) "Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery" Analytical Biochemistry 332(1):153-9.:10.1016 / j.ab.2004.04.031, PMID 15301960 with the following modifications. A 50 μl reaction in 50 mM sodium acetate, 5X SYPRO Orange and enzyme supernatant at pH 6 was subjected to a thermal ramp from 50°C to 98°C at 1°C per minute. Dye fluorescence was monitored using a BioRad CFX 384 real-time PCR machine. Melt curve data were analyzed using the supplied CFX Manager software.

[0114] Example 4: Expression of variant β-amylase

[0115] By constructing an expression plasmid containing the coding polynucleotide sequence, the plasmid is transformed into methanol yeast (Phaffi's yeast), and the resulting expression strain is grown in the following manner to obtain a variant polypeptide with beta-amylase activity. The fresh methanol yeast cells of the expression strain are obtained by spreading the glycerol stock of the sequence-confirmed strain onto a yeast extract peptone dextrose (YPD) agar plate containing zeocin. After 2 days, the starting seed culture of the production strain is inoculated into 100mL buffered glycerol composite medium (BMGY) using cells from these plates, and grown for 20-24 hours at 30°C and 225-250rpm. The scale of the seed culture is expanded by transferring a suitable amount to a 2-4L BMMY medium in a fermenter equipped with baffles. Fermentation is carried out for 48-72 hours at 30°C and under the stirring of 1100rrp provided by flat blade impellers. After the initial batch stage of fermentation, sterile filtered methanol was added as feed whenever the dissolved oxygen level in the culture dropped below 30%. Alternatively, feed was added every 3 hours at 0.5% v / v of the starting batch culture. The final fermentation broth was centrifuged at 7000×g for 30 minutes at 4°C to obtain a cell-free supernatant.

[0116] Variant polypeptides with β-amylase activity are identified by analyzing supernatants for relevant protein expression by SDS-PAGE or capillary electrophoresis.

[0117] Example 5: Mutant β-amylase

[0118] The parent enzyme is selected from KITAMOTO, "Cloning and Sequencing of the Gene Encoding Thermophilic B-amylase of Clostridium Thermosulfurogenes" (1988) J. Bacteriology, Vol. 170, pp. 5848-5854; NCBI_P19584.1 is AAA23204.1, and the amino acid sequence identified in the present application as SEQ ID NO: 1, which is encoded by the nucleic acid sequence SEQ ID NO.: 2. The parent enzyme is engineered in the laboratory to produce a non-naturally occurring β-amylase variant enzyme with improved enzyme characteristics. The improved characteristics include thermostability, pH, enzyme activity, or any combination thereof.

[0119] Variant polypeptide enzymes are formed starting with a parent enzyme and are evolved using gene site saturation mutagenesis (GSSM) of the parent enzyme as described in at least US6562594, US6171820 and US6764835, error-prone PCR and / or trimmed multi-site combinatorial assembly (TMSCA) as described in US Pat. No. 9476078.

[0120] When compared with the parent enzyme, the β-amylase variant enzyme can be an amino acid insertion, deletion or substitution. The substitution can be an amino acid residue of a different amino acid; the substitution can be a conservative amino acid substitution, or the substitution can be a similar amino acid residue. In addition, the substitution can be located at more than one site of the amino acid sequence. For example, the β-amylase variant can have at least two amino acid substitutions, wherein the substitution is located at amino acid residues 143 and 197. In addition, the parent enzyme can be substituted multiple times. For example, the β-amylase variant can have an amino acid substitution at amino acid residue numbers 203, 364, 369 and 398. Additional examples of structural changes in the amino acid sequence of the parent enzyme and a combination of single-point amino acid substitutions or amino acid substitutions and improved enzymatic activity, thermostability, pH and any combination thereof when compared with the parent enzyme are shown in Table 1 below.

[0121] Table 1:

[0122]

[0123]

[0124]

[0125] Example 6: pH of variant β-amylase

[0126] Variant polypeptides with β-amylase activity and pH profiles of the enzyme were determined using the BCA assay: substrate was 1% pullulan from potato (Sigma A8515 lot SLBS5819); reaction buffer was 50 mM Britton Robinson Buffer, 125 μL total; lyophilized enzyme; diluted enzyme 12.5 μL; wavelength reading 560 nm; pH 4.0, 4.5, 5.0, 5.5, 6.0, 8.0 at 60°C. Before starting the reaction, 10 PCR strips / tubes were pre-heated for 10 minutes in a thermal cycler. Incubate 112.5 μl (buffer) in PCR: 1% pullulan in any given buffer at the appropriate temperature for a minimum of 10 minutes. Initiate the reaction by adding 12.5 μL of enzyme dilution. At each time point (TP), a 10 uL aliquot of the reaction was placed in a BCA solution plate. Once all time points were taken, the BCA baffle was heated at 80°C for 30 minutes and cooled. 100 μL of the developed BCA solution was transferred to a 96-well flat bottom plate and read at 560 nm. The results are shown in Table 2 and Figure 1 middle.

[0127] Table 2:

[0128]

[0129]

[0130]

[0131] Example 7: Baking wheat bread

[0132] The baking performance of the variant polypeptides having β-amylase activity was tested in wheat bread produced by the direct method. 1000 g of 550 type flour (Vogtmühlen Illertissen), 30 g of compressed yeast, 20 g of salt, 20 g of sugar, 20 g of margarine, 60 ppm of ascorbic acid, 150 ppm of CS 30 (fungal xylanase, cellulase, fungal α-amylase), 8g 55 (distilled monoglyceride) and 580 g of water were mixed in a Kemper SP 15 spiral mixer at speed 1 for 4.5 minutes and at speed 2 for 2.25 minutes until the final dough temperature was 28°C. After resting for 15 minutes, the dough was divided into 500 g pieces, rounded and proofed for 15 minutes. After that, the dough pieces were molded, placed in baking tins and proofed for 80 minutes at 35°C and 85% relative humidity. The proofed dough pieces were baked in a cabinet oven at 255°C / 240°C under lower and upper heating for 25 minutes and steam injection for 15 seconds.

[0133] The variant peptide enzyme samples were added to flour at dosages ranging from 27 ppm to 325 ppm. The effects on dough properties and final baked products were compared with the negative control (no enzyme), Nonovayl 10,000 and Novamyl 3D.

[0134] The volume effect was determined by measuring the bread loaves by a laser scanner (Stable Micro Systems VolScan Profiler, VolScan 600). The negative control was defined as 0%. The dough properties were tactilely assessed by a skilled senior baker and described in comparison with the negative control.

[0135] The prepared baked bread was packaged and sealed in plastic bags. In addition, it was partially pasteurized at 85°C for 90 minutes. Freshly and after a defined storage time, typically after 1, 10, 20 days, the effect on the crumb properties was determined by texture profile analysis using a texture analyzer (Stable Micro Systems, TA.XTplus Texture Analyzer). Therefore, before the measurement, a 25 mm thick slice was cut from the middle of the loaf. The results are shown in Table 3 and Figure 2 as shown in .

[0136]

Claims

1. A variant polypeptide having beta-amylase activity, wherein the variant polypeptide is encoded by a nucleic acid sequence that is at least 80% identical, 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%, at least 99% or at least 100% identical to the nucleic acid sequence as set forth in SEQ ID NO:

1.

2. A variant polypeptide having β-amylase activity, wherein the variant polypeptide is selected from the group consisting of: (a) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, wherein the variant polypeptide has an amino acid residue numbering at the following position to that of SEQ ID NO: 16, 19, 24, 25, 27, 28, 48, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or any combination thereof to the amino acid sequence of SEQ ID NO: 2, and the variant polypeptide has β-amylase activity; (b) the variant polypeptide is ID NO:3 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:3, wherein the variant polypeptide has the amino acid residue numbering at the following position to SEQ ID NO: at least one single amino acid modification of the amino acid sequence of NO:3: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:3 Any combination of the amino acid sequences of NO:3, and the variant polypeptide has β-amylase activity;(b) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:4, wherein the variant polypeptide has an amino acid sequence at the following amino acid residue position numbering to that of SEQ ID NO:4: at least one single amino acid modification of the amino acid sequence of NO:4: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:4 or any combination thereof, and the variant polypeptide has beta-amylase activity; (d) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:5, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:4 at the following amino acid residue position number: at least one single amino acid modification of the amino acid sequence of SEQ ID NO:5: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:5 Any combination of the amino acid sequence of NO:5, and the variant polypeptide has β-amylase activity;(e) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:6, wherein the variant polypeptide has an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:6, at least one single amino acid modification of the amino acid sequence of NO:6: 16, 19, 24, 25, 27, 28, 48, 83, 101, 105, 107, 119, 141, 143, 145, 146, 147, 153, 155, 172, 175, 179, 197, 201, 203, 205, 206, 215, 219, 220, 221, 230, 273, 280, 286, 319, 322, 364, 366, 369, 398, 399, 438, 440, 446, 452, 453, 456, 458, 463, 464, 465, 468, 476, 490, 499, 504, 508, or to SEQ ID NO:6, and the variant polypeptide has beta-amylase activity; and (f) the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:7, wherein the variant polypeptide has a residue number corresponding to SEQ ID NO:7 at the following amino acid residue position numbering. at least one single amino acid modification of the amino acid sequence of NO:7: 48, 51, 56, 57, 59, 60, 80, 115, 133, 137, 139, 151, 173, 175, 177, 178, 179, 185, 187, 204, 207, 211, 229, 233, 235, 237, 238, 247, 251, 252, 253, 262, 305, 231, 318, 351, 354, 396, 398, 401, 430, 431, 470, 472, 478, 484, 485, 488, 490, 495, 496, 497, 500, 508, 522, 531, 536, 540, or to SEQ ID NO:7 Any combination of the amino acid sequence of NO:7, and the variant polypeptide has β-amylase activity. ; 3. The variant polypeptide of claim 2, wherein the at least one single amino acid modification is an amino acid substitution, insertion, deletion, or any combination thereof, and the variant polypeptide has β-amylase activity.

4. The variant polypeptide according to claim 3, wherein the amino acid substitution is a conservative amino acid substitution.

5. The variant polypeptide of claim 3, wherein the at least one single amino acid modification is an amino acid substitution comprising the following: K16Q, D19I, D19L, K24D, K24E, D25P, L27Q, L27H, L27C, I28A, K48Q, E48D, D51I, D51L, K56D, K56E, D57P, D57K, L59Q, L59H, L59C, I60A, E80D, C83S, S83C, T101N, Q105L, N107S, C115S, S115C, N119D, T133N, Q137L, N139S, A141R, N143D, S14 5N, S146P, Y147H, N151D, K153E, Y155H, H155Y, S172T, A173R, N175D, W1 75R, S177N, S178P, G179D, Y179H, K185E, Y187H, H187Y, A197T, K201E, G 203I, S204T, I205M, A206H, W207R, G211D, S215D, F219W, S220W, S220C, C220S, C220L, C220W, Q221M, A229T, N230K, K233E, G235I, I237M, A238H, S247D, F251W, S252L, S252W, S252C, C252S, C252L, C252W, Q253M, N262K , N262G, V273*, A280S, S280A, H286Y, Y286H, V305*, A312S, S312A, Y318 H, H318Y, T319S, S319T, C322S, S322C, T351S, S351T, C354S, S354C, A36 4P, S366H, N369P, A396P, S398H, S398P, D399M, N401P, S430P, D431M, N43 8Y, N738S, Y440N, S446P, N452D, T453K, A456S, G458D, P463T, P463L, N464D, Y465N, W468C, N470Y, N470S, Y472N, P476L, S478P, N484D, T485K, A488S, G490D, S490T, P495T, P495L, N496D, Y497N, S499P, W500C, T504N, P508L, S508T, S522T, S531P, T536N, S540T, or any combination thereof, and the variant polypeptide has β-amylase activity.

6. A variant polypeptide having β-amylase activity, wherein the variant polypeptide is selected from the group consisting of: (i) the variant polypeptide is an amino acid sequence that is at least 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, wherein the variant polypeptide has a combination of amino acid modifications to the amino acid sequence of SEQ ID NO:2, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; <h2 style=";text-align:left;direction:ltr">(b1)D19I、L27C、I28A、S145N、G203I、S220L、A364P、N369P、S398P;(c1)D19I、L27H、Q221M;(d1)D25K、A364P、N369P、S398P;(e1)D25K、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr"> (f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、S220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr"> (j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1)D25K、L27C、I28A、A141R、A364P、N369P、S398P;(l1)D25K、L27C、I28A、A141R、A364P、N369 P、S398P;(m1)D25K、L27C、S145N、S220L、A364P、N369P、S398P;(n1)D25K、 L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203 I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P;(r1)K24E、D 25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141 R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、A364P、N369P、S398 P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M; <h2 style=";text-align:left;direction:ltr">(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W46 8C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y440N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364 P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h 2) D25K, A364P, N369P, S398P, N452D;(i2) D25K, A364P, N369P, S398P, N438S;(j2) D25K, A364P, N369P, S398P, T453K;(k2) D25K, A364P, N369P, S39 8P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, S220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N3 69P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K、L27C、S220L、A364P、N 369P、S398P、N107S、S172T、W175R;(p2)D25K、L27C、S220L、A364P、N369P、 S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398P, P463T;(r2)D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2 (t2)D25K,L27C,S220L,A364P,N369P,S398P,P463L,Y465N,W468C(u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (ii) the variant polypeptide is the same as SEQ ID NO:3 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:3, wherein the variant polypeptide has a combination of amino acid modifications to the amino acid sequence of SEQ ID NO:3, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; <h2 style=";text-align:left;direction:ltr"> (b1)D19I、L27C、I28A、S145N、G203I、S220L、A364P、N369P、S398P;(c1)D19I、L27H、Q221M;(d1)D25K、A364P、N369P、S398P;(e1)D25K、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr">(f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、S220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr"> (j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1)D25K、L27C、I28A、A141R、A364P、N369P、S398P;(l1)D25K、L27C、I28A、A141R、A364P、N369 P、S398P;(m1)D25K、L27C、S145N、S220L、A364P、N369P、S398P;(n1)D25K、 L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203 I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P;(r1)K24E、D 25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141 R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、A364P、N369P、S398 P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M; <h2 style=";text-align:left;direction:ltr">(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W46 8C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y440N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364 P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h 2) D25K, A364P, N369P, S398P, N452D;(i2) D25K, A364P, N369P, S398P, N438S;(j2) D25K, A364P, N369P, S398P, T453K;(k2) D25K, A364P, N369P, S39 8P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, S220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N3 69P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K、L27C、S220L、A364P、N 369P、S398P、N107S、S172T、W175R;(p2)D25K、L27C、S220L、A364P、N369P、 S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398P, P463T;(r2)D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2 (t2)D25K,L27C,S220L,A364P,N369P,S398P,P463L,Y465N,W468C(u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (iii) the variant polypeptide is the same as SEQ ID NO:4 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:4, wherein the variant polypeptide has a combination of amino acid modifications to the amino acid sequence of SEQ ID NO:4, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; <h2 style=";text-align:left;direction:ltr"> (b1)D19I、L27C、I28A、S145N、G203I、S220L、A364P、N369P、S398P;(c1)D19I、L27H、Q221M;(d1)D25K、A364P、N369P、S398P;(e1)D25K、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr"> (f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、S220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr">(j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1)D25K、L27C、I28A、A141R、A364P、N369P、S398P;(l1)D25K、L27C、I28A、A141R、A364P、N369 P、S398P;(m1)D25K、L27C、S145N、S220L、A364P、N369P、S398P;(n1)D25K、 L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203 I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P;(r1)K24E、D 25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141 R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、A364P、N369P、S398 P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M; <h2 style=";text-align:left;direction:ltr">(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W46 8C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y440N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364 P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h 2) D25K, A364P, N369P, S398P, N452D;(i2) D25K, A364P, N369P, S398P, N438S;(j2) D25K, A364P, N369P, S398P, T453K;(k2) D25K, A364P, N369P, S39 8P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, S220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N3 69P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K、L27C、S220L、A364P、N 369P、S398P、N107S、S172T、W175R;(p2)D25K、L27C、S220L、A364P、N369P、 S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398P, P463T;(r2)D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2 (t2)D25K,L27C,S220L,A364P,N369P,S398P,P463L,Y465N,W468C(u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (iv) the variant polypeptide is the same as SEQ ID NO:5 is at least 80% identical, 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% identical to the amino acid sequence set forth in NO:5, wherein the variant polypeptide has a sequence identical to SEQ ID NO:5 amino acid sequence of the combination of modifications, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; (b1) D19I, L27C, I28A, S145N, G203I, C220L, A364P, N369P, S398P; (c1) D19I, L27H, Q221M; (d1) D25K, A364P, N369P, S398P; (e1) D25K, A364P, N369P, S398P; <h2 style=";text-align:left;direction:ltr"> (f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、C220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr">(j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1)D25K、L27C、I28A、A141R、A364P、N369P、S398P;(l1)D25K、L27C、I28A、A141R、A364P、N369 P、S398P;(m1)D25K、L27C、S145N、C220L、A364P、N369P、S398P;(n1)D25K、 L27C、C220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203 I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P;(r1)K24E、D 25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141 R、G203I、C220L、S398P;(t1)K24E、L27H、I28A、C220L、A364P、N369P、S398 P;(u1)N143D、A197T;(v1)C220L、A364P、N369P、S398P;(w1)T101N、D399M; <h2 style=";text-align:left;direction:ltr">(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W46 8C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y440N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364 P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h 2) D25K, A364P, N369P, S398P, N452D;(i2) D25K, A364P, N369P, S398P, N438S;(j2) D25K, A364P, N369P, S398P, T453K;(k2) D25K, A364P, N369P, S39 8P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, C220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, C220L, A364P, N3 69P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K、L27C、C220L、A364P、N 369P、S398P、N107S、S172T、W175R;(p2)D25K、L27C、C220L、A364P、N369P、 S398P, Q105L, N119D;(q2)D25K, L27C, C220L, A364P, N369P, S398P, P463T;(r2)D25K, L27C, C220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2 (t2)D25K,L27C,C220L,A364P,N369P,S398P,P463L,Y465N,W468C(u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (vi) the variant polypeptide is the same as SEQ ID NO:6 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:6, wherein the variant polypeptide has a combination of amino acid modifications to the amino acid sequence of SEQ ID NO:6, and the combination is selected from the group consisting of: (a1) D19I, L27C, A141R, A364P, N369P, S398P; <h2 style=";text-align:left;direction:ltr"> (b1)D19I、L27C、I28A、S145N、G203I、S220L、A364P、N369P、S398P;(c1)D19I、L27H、Q221M;(d1)D25K、A364P、N369P、S398P;(e1)D25K、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr"> (f1)D25K、G203I、A364P、N369P、S398P;(g1)D25K、I28A、S220L、Q221M、A364P、N369P;(h1)D25K、L27C、G203I、A364P、N369P、S398P;(i1)D25K、L27C、G203I、A364P、N369P、S398P; <h2 style=";text-align:left;direction:ltr">(j1)D25K、L27C、G203I、Q221M、A364P、N369P、S398P;(k1)D25K、L27C、I28A、A141R、A364P、N369P、S398P;(l1)D25K、L27C、I28A、A141R、A364P、N369 P、S398P;(m1)D25K、L27C、S145N、S220L、A364P、N369P、S398P;(n1)D25K、 L27C、S220L、A364P、N369P、S398P;(o1)D25P、L27H、I28A、Q221M;(p1)G203 I、A364P、N369P、S398P;(q1)K24E、D25K、L27C、A364P、N369P;(r1)K24E、D 25K、L27C、I28A、A141R、A364P、N369P、S398P;(s1)K24E、D25P、L27H、A141 R、G203I、S220L、S398P;(t1)K24E、L27H、I28A、S220L、A364P、N369P、S398 P;(u1)N143D、A197T;(v1)S220L、A364P、N369P、S398P;(w1)T101N、D399M; <h2 style=";text-align:left;direction:ltr">(X1)D25K、A364P、N369P、S398P、P463T;(y1)D25K、A364P、N369P、S398P、N438Y、N464D、P476L;(z1)D25K、A364P、N369P、S398P、P463L、Y465N、W46 8C;(a2)D25K、A364P、N369P、S398P、A456S;(b2)D25K、A364P、N369P、S398P、N438Y、Y440N;(c2)D25K、A364P、N369P、S398P、G458D;(d2)D25K、A364 P, N369P, S398P, S508T;(e2)D25K, A364P, N369P, S398P, T504N;(f2)D25K, A364P, N369P, S398P, S490T;(g2)D25K, A364P, N369P, S398P, S499P;(h 2) D25K, A364P, N369P, S398P, N452D;(i2) D25K, A364P, N369P, S398P, N438S;(j2) D25K, A364P, N369P, S398P, T453K;(k2) D25K, A364P, N369P, S39 8P, S446P;(l2)D25K, A364P, N369P, S398P, W468C;(m2)D25K, L27C, S220L, A364P, N369P, S398P, K153E, K201E;(n2)D25K, L27C, S220L, A364P, N3 69P、S398P、E48D、N143Y、S146P、K201E;(o2)D25K、L27C、S220L、A364P、N 369P、S398P、N107S、S172T、W175R;(p2)D25K、L27C、S220L、A364P、N369P、 S398P, Q105L, N119D;(q2)D25K, L27C, S220L, A364P, N369P, S398P, P463T;(r2)D25K, L27C, S220L, A364P, N369P, S398P, N438Y, N464D, P476L;(s2 (t2)D25K,L27C,S220L,A364P,N369P,S398P,P463L,Y465N,W468C(u2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, P463T; (v2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, N438Y, N464D, P476L; and (w2) D25K, L27C, G203I, Q221M, A364P, N369P, S398P, S398P, P463L, Y465N, W468C; wherein the variant polypeptide has β-amylase activity; (vii) the variant polypeptide is the same as SEQ ID NO:7 is at least 80% identical, 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% identical to the amino acid sequence set forth in SEQ ID NO:7, wherein the variant polypeptide has a sequence identical to SEQ ID NO:7 amino acid sequence, and the combination is selected from the group consisting of: (a1) D51I, L59C, A173R, A396P, N401P, S430P; (b1) D51I, L59C, I60A, S177N, G235I, S252L, A396P, N401P, S430P; (c1) D51I, L59H, Q253M; (d1) D57K, A396P, N401P, S430P; (e1) D57K, A396P, N401P, S430P; (f1) D57K, G235I, A396P, N401P, S430P; (g1) D57K, I60A, S252L, Q253M, A396P, N401P; (h1) D57K, L59C, G235I, A396P, N401P, S 430P; (i1) D57K, L59C, G235I, A396P, N401P, S430P; (j1) D57K, L59C, G235I, Q253M, A396P, N4 01P, S430P; (k1) D57K, L59C, I60A, A173R, A396P, N401P, S430P; (l1) D57K, L59C, I60A, A173R , A396P, N401P, S430P; (m1) D57K, L59C, S177N, S252L, A396P, N401P, S430P; (n1) D57K, L59C, S252L, A396P, N401P, S430P; (o1) D57P, L59H, I60A, Q253M; (p1) G235I, A396P, N401P, S430P;<h2 style=";text-align:left;direction:ltr">(q1)K56E、D57K、L59C、A396P、N369P;(r1)K56E、D57K、L59C、I60A、A173R、A396P、N401P、S430P;(s 1)K56E、D57P、L59H、A173R、G235I、S252L、S430P;(t)K56E、L59H、I60A、S252L、A396P、N401P、S430 P;(u1)N175D、A229T;(v1)S252L、A396P、N401P、S430P;(w1)T133N、D431M;(x1)D57K、A396P、N401 P、S430P、P495T;(y1)D57K、A396P、N401P、S430P、N470Y、N496D、P508L;(z1)D57K、A396P、N401P、S4 30P, P495L, Y497N, W500C;(a2)D57K, A396P, N401P, S430P, A488S;(b2)D57K, A396P, N401P, S430P, N470Y, Y472N;(c2)D57K, A396P, N401P, S430P, G490D;(d2)D57K, A396P, N401P, S430P, S540T;(e 2) D57K, A396P, N401P, S430P, T540N;(f2) D57K, A396P, N401P, S430P, S522T;(g2) D57K, A396P, N401P, S430P, S531P;(h2) D57K, A396P, N401P, S430P, N484D;(i2) D57K, A396P, N401P, S430P, N470S; (j2)D57K, A396P, N401P, S430P, T485K; (k2) D57K, A396P, N401P, S430P, S478P; (l2) D57K, A396P, N401P, S430 P, W500C; (m2) D57K, L59C, S252L, A396P, N401P, S430P, K185E, K233E; (n2) D57K, L59C, S252L, A396P, N401P, S4 30P, E48D, N175Y, S178P, K233E; (o2) D57K, L59C, S252L, A396P, N401P, S430P, N139S, S204T, W207R; (p2) D57K , L59C, S252L, A396P, N401P, S430P, Q137L, N151D; (q2) D57K, L59C, S252L, A396P, N401P, S430P, P495T; (r2) D5 7K, L59C, S252L, A396P, N401P, S430P, N470Y, N496D, P508L; (s2) D57K, L59C, S252L, A396P, N401P, S430P, P49 5L, Y497N, W500C; (t2) D57K, L59C, K185E, K233E, S252L, A396P, N401P, S430P, N470Y, N496D, P508L; (u2) D57K, L59C, G235I, Q253M, A396P, N401P, S430P, P495T; (v2) D57K, L59C, G235I, Q253M, A396P, N401P, S430P, N470Y, N496D, P508L; (w2) D57K, L59C, G235I, Q253M, A396P, N401P, S430P, P495L, Y497N, W500C; wherein the variant polypeptides have β-amylase activity.

7. A variant polypeptide having β-amylase activity, wherein the variant polypeptide is an amino acid sequence that is at least 80% identical, 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% identical to the amino acid sequence as set forth in SEQ ID NO:2, and the variant polypeptide has increased enzyme activity, thermal stability, pH stability or any combination thereof when compared to the β-amylase of SEQ ID NO:

2.

8. The variant polypeptide according to any one of claims 1 to 7, wherein the variant polypeptide is a fragment of the full-length amino acid sequence, and the fragment has β-amylase activity.

9. A variant polypeptide comprising a hybrid of at least one variant polypeptide according to any one of claims 1 to 7 and a second polypeptide having amylase activity, wherein the hybrid has β-amylase activity.

10. A composition comprising the variant polypeptide according to any one of claims 1 to 7.

11. A composition comprising the variant polypeptide according to any one of claims 1 to 7 and a second enzyme.

12. The composition of claim 11, wherein the second enzyme is selected from the group consisting of a second β-amylase, a lipase, an α-amylase, a G4-amylase, a xylanase, a protease, a cellulase, a glucoamylase, an oxidoreductase, a phospholipase, and a cyclodextrin glucanotransferase.

13. A method for making a variant polypeptide, comprising: providing a template nucleic acid sequence encoding a polypeptide variant according to any one of claims 1 to 7, transforming the template nucleic acid sequence into an expression host, cultivating the expression host to produce the variant polypeptide, and purifying the variant polypeptide.

14. The method of claim 13, wherein the template nucleic acid is a variant nucleotide of the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide is a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence as set forth in SEQ ID NO: 1, wherein the variant nucleotide encodes a polypeptide having β-amylase activity.

15. The method of claim 13, wherein the expression host is selected from the group consisting of a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.

16. The method according to claim 15, wherein the bacterial expression system is selected from the group consisting of E. coli, Bacillus, Pseudomonas and Streptomyces.

17. The method according to claim 15, wherein the yeast expression system is selected from Candida, Pichia, Saccharomyces, Schizosaccharomyces.

18. The method of claim 15, wherein the fungal expression system is selected from the group consisting of Penicillium, Aspergillus, Fusarium, Myceliopthora, Rhizomucor, Rhizopus, Thermomyces, and Trichoderma.

19. A method of preparing a dough or a baked product prepared from said dough, said method comprising adding one of the variant polypeptides according to any one of claims 1 to 7 to said dough, and baking it.

20. A method of using the variant polypeptide according to any one of claims 1 to 7 for treating starch.

21. A method of using a variant polypeptide according to any one of claims 1 to 7 for cleaning or washing fabrics, hard surfaces or dishes.

22. A method of using the variant polypeptide according to any one of claims 1 to 7 for producing ethanol.

23. A method of using the variant polypeptide according to any one of claims 1 to 7 for treating an oil well.

24. A method of using a variant polypeptide according to any one of claims 1 to 7 for treating pulp or paper.

25. A method of using the variant polypeptide according to any one of claims 1 to 7 for feeding an animal.

26. A method of using the variant polypeptide according to any one of claims 1 to 7 for preparing a syrup.

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