Method for asymmetric reduction of prochiral olefins using alkene reductase or mutants thereof

Abstract

The invention discloses an asymmetric reduction method for prochiral olefin by using alkene reductase or a mutant thereof. Specifically, the invention discloses a preparation method of a compound as shown in a formula I. The preparation method comprises the following step: in a solvent, in the presence of alkene reductase or a mutant thereof and coenzyme, a compound as shown in a formula II is subjected to a reduction reaction as shown in the specification, and the compound as shown in the formula I is prepared. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a method for asymmetric reduction of prochiral olefins using enoate reductase or its mutants. Background Art

[0002] Chiral chroman compounds are intermediates of important drugs in clinical practice. The current synthesis method is to use ruthenium as a metal catalyst and a chiral ligand to catalyze the asymmetric reduction of double bonds, and then rely on chemical resolution to obtain them. The atom economy is low, the price is high, and the yield is very low. Existing commercial enzymes and natural enzymes cannot produce the required configuration.

[0003] Therefore, there is an urgent need in the art for an enoate reductase with high stereoselectivity and a method for asymmetric reduction of prochiral olefins using the same. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defect of insufficient types of preparation methods of chiral chroman compounds in the prior art, and provide a new method for asymmetric reduction of prochiral olefins using enoate reductase and its mutants. The asymmetric reduction reaction of olefins catalyzed by the enoate reductase in the present invention has one or more of the following advantages: high raw material conversion rate, high yield, high chiral purity of the product, high stereoselectivity, and mild conditions.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a method for preparing a compound represented by formula I, which comprises the following steps: in a solvent, in the presence of enoate reductase or its mutant and a coenzyme, the compound represented by formula II undergoes the following reduction reaction to prepare the compound represented by formula I,

[0007]

[0008] wherein, R 1 is methyl, ethyl, n-propyl, isopropyl or hydroxyl; R 2 is methyl, ethyl, n-propyl, aldehyde group, hydroxyl or carboxyl;

[0009] The coenzyme is the coenzyme of the enoate reductase or its mutant;

[0010] The amino acid sequence of the enoate reductase is as shown in SEQ ID NO:1;

[0011] The mutant has differences in amino acid residues at one or more sites selected from the following positions in the amino acid sequence shown in SEQ ID NO: 1: position 2, position 3, position 4, position 5, position 8, position 9, position 10, position 12, position 13, position 17, position 19, position 20, position 21, position 23, position 24, position 26, position 27, position 28, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 46, position 51, position 53, position 55, position 56, position 57, position 58, position 59, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 71, position 73, position 74, position 75, position 76, position 77, position 80, position 81, position 82, position 83, position 85, position 86, position 87, position 88, position 89, position 92, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 128, position 135, position 136, position 137, position 145, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 167, position 168, position 169, position 170, position 172, position 173, position 174, position 175, position 177, position 179, position 180, position 181, position 184, position 189, position 190, position 191, position 192, position 193, position 194, position 195, position 196, position 197, position 198, position 199, position 200, position 201, position 207, position 208, position 209, position 210, position 211, position 212, position 213, position 214, position 215, position 216, position 217, position 218, position 219, position 221, position 222, position 223, position 224, position 225, position 226, position 227, position 228, position 229, position 231, position 232, position 233, position 234, position 242, position 243, position 244, position 245, position 246, position 247, position 248The 249th, 250th, 251st, 252nd, 253rd, 254th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 277th, 278th, 283rd, 284th, 285th, 287th, 288th, 289th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 320th, 321st, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 338th, 339th, 340th, 341st, 342nd, 343rd, 344th, 346th, 349th, 350th, 351st, 352nd, 353rd, 354th, 356th, 357th, 361st, 362nd, 363rd, 367th, 368th, 369th, 370th, 371st, 372nd, 373rd, 375th, 376th, 377th, 379th, 380th, 382nd, 383rd, 384th, 386th, 388th, 389th, 390th, 393rd, 395th and 396th.

[0012] In one embodiment, the difference is a deletion, an addition or a substitution, preferably a substitution.

[0013] In one embodiment, the mutant has the amino acid residue at position 2 of the amino acid sequence shown in SEQ ID NO: 1 replaced by K, N or V, and / or the amino acid residue at position 3 replaced by G, I or K, and / or the amino acid residue at position 4 replaced by A, D or E, and / or the amino acid residue at position 5 replaced by E or G, and / or the amino acid residue at position 8 replaced by A or K, and / or the amino acid residue at position 9 replaced by D, and / or the amino acid residue at position 10 replaced by M, N or V, and / or, and / or, the amino acid residue at position 12 replaced by M, Q, R or T, and / or the amino acid residue at position 13 replaced by D or V, and / or the amino acid residue at position 17 replaced by C, N or Y, and / or the amino acid residue at position 19 replaced by A, and / or the amino acid residue at position 20 replaced by D, and / or the amino acid residue at position 21 replaced by E, F or K, and / or the amino acid residue at position 23 replaced by W, and / or the amino acid residue at position 24 replaced by D, R or T, and / or the amino acid residue at position 26 replaced by Q or W, and / or the amino acid residue at position 27 replaced by H or T, and / or the amino acid residue at position 28 replaced by G or W, and / or the amino acid residue at position 31 replaced by I or W, and / or the amino acid residue at position 32 replaced by N or Y, and / or the amino acid residue at position 33 replaced by H or T, and / or the amino acid residue at position 34 replaced by C, L, V or W, and / or the amino acid residue at position 35 replaced by F, M, T or Y, and / or the amino acid residue at position 36 replaced by M, and / or the amino acid residue at position 37 replaced by G, and / or the amino acid residue at position 38 replaced by D, and / or the amino acid residue at position 39 replaced by E, G, I, P or T, and / or the amino acid residue at position 40 replaced by A or D, and / or the amino acid residue at position 41 replaced by A, D, L or Q, and / or the amino acid residue at position 42 replaced by G or I, and / or the amino acid residue at position 43 replaced by C, E, G, P or V, and / or the amino acid residue at position 44 replaced by W or Y, and / or the amino acid residue at position 46 replaced by E or I, and / or the amino acid residue at position 51 replaced by N, and / or the amino acid residue at position 53 replaced by E, G or I, and / or the amino acid residue at position 55 replaced by C, G, R or T, and / or the amino acid residue at position 56 replaced by I, K, R or T, and / or the amino acid residue at position 57 replaced by I, K, M or W, and / or the amino acid residue at position 58 replaced by G or N, and / or the amino acid residue at position 59 replaced by F, M or V, and / or the amino acid residue at position 61 replaced by C or Y, and / or the amino acid residue at position 62 replaced by M or V, and / or the amino acid residue at position 63 replaced by Y, and / or the amino acid residue at position 64 replaced by D or I, and / or the amino acid residue at position 65 replaced by C, D or W, and / or the amino acid residue at position 66 replaced by L, and / orThe 67th amino acid residue is replaced with L or N, and / or, the 68th amino acid residue is replaced with P or Q, and / or, the 69th amino acid residue is replaced with E, and / or, the 71st amino acid residue is replaced with G or V, and / or, the 73rd amino acid residue is replaced with A, Q or V, and / or, the 74th amino acid residue is replaced with W, and / or, the 75th amino acid residue is replaced with C, E, L or S, and / or, the 76th amino acid residue is replaced with N or V, and / or, the 77th amino acid residue is replaced with R, V or Y, and / or, the 80th amino acid residue is replaced with K or N, and / or, the 81st amino acid residue is replaced with C, I or T, and / or, the 82nd amino acid residue is replaced with D or T, and / or, the 83rd amino acid residue is replaced with A, F, H, I or M, and / or, the 85th amino acid residue is replaced with G or L, and / or, the 86th amino acid residue is replaced with R or S, and / or, the 87th amino acid residue is replaced with R or W, and / or, the 88th amino acid residue is replaced with C, F, K or R, and / or, the 89th amino acid residue is replaced with G, T or W, and / or, the 92nd amino acid residue is replaced with H, P or W, and / or, the 95th amino acid residue is replaced with L or R, and / or, the 96th amino acid residue is replaced with E, and / or, the 97th amino acid residue is replaced with P or R, and / or, the 98th amino acid residue is replaced with A, I, K, M, N or Y, and / or, the 99th amino acid residue is replaced with I, N or R, and / or, the 100th amino acid residue is replaced with D, E or K, and / or, the 101st amino acid residue is replaced with F or H, and / or, the 102nd amino acid residue is replaced with P, T or V, and / or, the 103rd amino acid residue is replaced with A or E, and / or, the 104th amino acid residue is replaced with L or S, and / or, the 105th amino acid residue is replaced with E, and / or, the 106th amino acid residue is replaced with I, K or W, and / or, the 113th amino acid residue is replaced with A, E, I or Q, and / or, the 114th amino acid residue is replaced with Q, R or T, and / or, the 115th amino acid residue is replaced with M or R, and / or, the 116th amino acid residue is replaced with H or W, and / or, the 117th amino acid residue is replaced with N, and / or, the 118th amino acid residue is replaced with E, and / or, the 119th amino acid residue is replaced with E or Y, and / or, the 120th amino acid residue is replaced with I, and / or, the 121st amino acid residue is replaced with D, F, K or N, and / or, the 122nd amino acid residue is replaced with R, and / or, the 123rd amino acid residue is replaced with E, F, K or P, and / or, the 124th amino acid residue is replaced with H, I or T, and / or, the 125th amino acid residue is replaced with M, Q or S, and / or, the 126th amino acid residue is replaced with A, G, I or L, and / or, the 128th amino acid residue is replaced with W, and / or, the 135th amino acid residue is replaced with Vand / or, the 136th amino acid residue is replaced with A or G, and / or, the 137th amino acid residue is replaced with K, and / or, the 145th amino acid residue is replaced with L, and / or, the 148th amino acid residue is replaced with A, F, H or V, and / or, the 149th amino acid residue is replaced with F, and / or, the 150th amino acid residue is replaced with F, K, N or P, and / or, the 151st amino acid residue is replaced with N, and / or, the 152nd amino acid residue is replaced with E, and / or, the 153rd amino acid residue is replaced with D, N or S, and / or, the 154th amino acid residue is replaced with L or T, and / or, the 155th amino acid residue is replaced with I or Y, and / or, the 156th amino acid residue is replaced with F, and / or, the 157th amino acid residue is replaced with L or W, and / or, the 158th amino acid residue is replaced with C, Q, R or T, and / or, the 159th amino acid residue is replaced with H, I or P, and / or, the 160th amino acid residue is replaced with L, and / or, the 161st amino acid residue is replaced with W, and / or, the 162nd amino acid residue is replaced with K or W, and / or, the 167th amino acid residue is replaced with E, P or S, and / or, the 168th amino acid residue is replaced with F, H, I or Y, and / or, the 169th amino acid residue is replaced with E, I or T, and / or, the 170th amino acid residue is replaced with C, H, K, M, N or P, and / or, the 172nd amino acid residue is replaced with L, and / or, the 173rd amino acid residue is replaced with I, and / or, the 174th amino acid residue is replaced with E, H or R, and / or, the 175th amino acid residue is replaced with W, and / or, the 177th amino acid residue is replaced with F, S or W, and / or, the 179th amino acid residue is replaced with F, G, P, T or V, and / or, the 180th amino acid residue is replaced with I or M, and / or, the 181st amino acid residue is replaced with F or Y, and / or, the 184th amino acid residue is replaced with F or P, and / or, the 189th amino acid residue is replaced with T or Y, and / or, the 190th amino acid residue is replaced with D, N or T, and / or, the 191st amino acid residue is replaced with F, K, L or R, and / or, the 192nd amino acid residue is replaced with E, G, I, M, R or S, and / or, the 193rd amino acid residue is replaced with F, L, M or T, and / or, the 194th amino acid residue is replaced with C, D, P or T, and / or, the 195th amino acid residue is replaced with E, H, K or Y, and / or, the 196th amino acid residue is replaced with R, and / or, the 197th amino acid residue is replaced with F or S, and / or, the 198th amino acid residue is replaced with Q, and / or, the 199th amino acid residue is replaced with F, and / or, the 200th amino acid residue is replaced with R, and / or, the 201st amino acid residue is replaced with M, and / or, the 207th amino acid residue is replaced with E, P or Q, and / or,The 208th amino acid residue is replaced with D, M, R, or V, and / or, the 209th amino acid residue is replaced with M, and / or, the 210th amino acid residue is replaced with D, H, or I, and / or, the 211th amino acid residue is replaced with A or R, and / or, the 212th amino acid residue is replaced with A, and / or, the 213th amino acid residue is replaced with H, V, or W, and / or, the 214th amino acid residue is replaced with M or N, and / or, the 215th amino acid residue is replaced with W or Y, and / or, the 216th amino acid residue is replaced with C or N, and / or, the 217th amino acid residue is replaced with M, R, or W, and / or, the 218th amino acid residue is replaced with C, F, L, P, or Q, and / or, the 219th amino acid residue is replaced with D or P, and / or, the 221st amino acid residue is replaced with C, E, K, W, or Y, and / or, the 222nd amino acid residue is replaced with K, R, V, or Y, and / or, the 223rd amino acid residue is replaced with H or L, and / or, the 224th amino acid residue is replaced with G, and / or, the 225th amino acid residue is replaced with A, and / or, the 226th amino acid residue is replaced with N, and / or, the 227th amino acid residue is replaced with D, and / or, the 228th amino acid residue is replaced with D, N, or R, and / or, the 229th amino acid residue is replaced with C, N, R, W, or Y, and / or, the 231st amino acid residue is replaced with K, Q, or S, and / or, the 232nd amino acid residue is replaced with A, D, G, N, or P, and / or, the 233rd amino acid residue is replaced with D, and / or, the 234th amino acid residue is replaced with H, I, M, or P, and / or, the 242nd amino acid residue is replaced with A, E, F, S, or W, and / or, the 243rd amino acid residue is replaced with A, F, H, or T, and / or, the 244th amino acid residue is replaced with Q, and / or, the 245th amino acid residue is replaced with A or V, and / or, the 246th amino acid residue is replaced with F, H, K, or Q, and / or, the 247th amino acid residue is replaced with C, W, or Y, and / or, the 248th amino acid residue is replaced with L, and / or, the 249th amino acid residue is replaced with W, and / or, the 250th amino acid residue is replaced with E, H, or Q, and / or, the 251st amino acid residue is replaced with A, D, R, or S, and / or, the 252nd amino acid residue is replaced with V, and / or, the 253rd amino acid residue is replaced with A, C, T, or Y, and / or, the 254th amino acid residue is replaced with G or V, and / or, the 256th amino acid residue is replaced with A, E, H, or V, and / or, the 257th amino acid residue is replaced with L, R, T, or V, and / or, the 258th amino acid residue is replaced with C or Q, and / or, the 259th amino acid residue is replaced with G, and / or, the 260th amino acid residue is replaced with D or Q, and / or, the 261st amino acid residue is replaced with D or Q, and / or, the 262nd amino acid residue is replaced with D, K, or Rand / or, the 263rd amino acid residue is replaced by C, and / or, the 264th amino acid residue is replaced by I, and / or, the 268th amino acid residue is replaced by I, M, P or S, and / or, the 269th amino acid residue is replaced by G or S, and / or, the 270th amino acid residue is replaced by K, and / or, the 271st amino acid residue is replaced by F or K, and / or, the 272nd amino acid residue is replaced by A, D or R, and / or, the 273rd amino acid residue is replaced by G, K, M or W, and / or, the 274th amino acid residue is replaced by G, M or P, and / or, the 277th amino acid residue is replaced by L, and / or, the 278th amino acid residue is replaced by C, and / or, the 283rd amino acid residue is replaced by E or F, and / or, the 284th amino acid residue is replaced by D, K or R, and / or, the 285th amino acid residue is replaced by H, S or W, and / or, the 287th amino acid residue is replaced by D, E, F, G, K, M or T, and / or, the 288th amino acid residue is replaced by A, F or R, and / or, the 289th amino acid residue is replaced by G or P, and / or, the 290th amino acid residue is replaced by I or Q, and / or, the 291st amino acid residue is replaced by A, and / or, the 292nd amino acid residue is replaced by E or G, and / or, the 293rd amino acid residue is replaced by F, K, N or P, and / or, the 294th amino acid residue is replaced by F, and / or, the 295th amino acid residue is replaced by L or N, and / or, the 296th amino acid residue is replaced by Q, and / or, the 297th amino acid residue is replaced by A, F, G, H, N or T, and / or, the 298th amino acid residue is replaced by E, and / or, the 299th amino acid residue is replaced by I, N or T, and / or, the 300th amino acid residue is replaced by P, Q or V, and / or, the 301st amino acid residue is replaced by C, and / or, the 302nd amino acid residue is replaced by H or N, and / or, the 303rd amino acid residue is replaced by F, T or V, and / or, the 304th amino acid residue is replaced by F, P or R, and / or, the 305th amino acid residue is replaced by H or K, and / or, the 309th amino acid residue is replaced by P, Q or S, and / or, the 310th amino acid residue is replaced by K or P, and / or, the 311th amino acid residue is replaced by A, E or Y, and / or, the 312th amino acid residue is replaced by E, H, K, L, M, V or Y, and / or, the 313th amino acid residue is replaced by E, P or T, and / or, the 314th amino acid residue is replaced by H, L or W, and / or, the 315th amino acid residue is replaced by G, P, V or Y, and / or, the 316th amino acid residue is replaced by E, F or S, and / or, the 317th amino acid residue is replaced by P, and / or, the 318th amino acid residue is replaced by F or R, and / or, the 320th amino acid residue is replaced by C, I or V, and / or, the 321st amino acid residue is replaced by E or Y, and / or,The 323rd amino acid residue is replaced by I, and / or, the 324th amino acid residue is replaced by E, M or T, and / or, the 325th amino acid residue is replaced by N, and / or, the 326th amino acid residue is replaced by L, and / or, the 327th amino acid residue is replaced by A, and / or, the 328th amino acid residue is replaced by P, S or Y, and / or, the 329th amino acid residue is replaced by A, F, M, N, Q or S, and / or, the 330th amino acid residue is replaced by R or W, and / or, the 331st amino acid residue is replaced by H, and / or, the 332nd amino acid residue is replaced by F, and / or, the 333rd amino acid residue is replaced by A or F, and / or, the 334th amino acid residue is replaced by D, E, F, P or W, and / or, the 335th amino acid residue is replaced by A, and / or, the 336th amino acid residue is replaced by P or Q, and / or, the 337th amino acid residue is replaced by I, P or V, and / or, the 338th amino acid residue is replaced by A, D or G, and / or, the 339th amino acid residue is replaced by L or T, and / or, the 340th amino acid residue is replaced by G or P, and / or, the 341st amino acid residue is replaced by E or T, and / or, the 342nd amino acid residue is replaced by C, P, Q, S or Y, and / or, the 343rd amino acid residue is replaced by C, K or M, and / or, the 344th amino acid residue is replaced by R or W, and / or, the 346th amino acid residue is replaced by N, and / or, the 349th amino acid residue is replaced by W, and / or, the 350th amino acid residue is replaced by K or S, and / or, the 351st amino acid residue is replaced by A, E, L, N or Q, and / or, the 352nd amino acid residue is replaced by M, and / or, the 353rd amino acid residue is replaced by N, Q or T, and / or, the 354th amino acid residue is replaced by Q, T or W, and / or, the 356th amino acid residue is replaced by H or I, and / or, the 357th amino acid residue is replaced by M, Q or R, and / or, the 361st amino acid residue is replaced by Y, and / or, the 362nd amino acid residue is replaced by K or W, and / or, the 363rd amino acid residue is replaced by D, G, I, P or Y, and / or, the 367th amino acid residue is replaced by H, N, R or V, and / or, the 368th amino acid residue is replaced by A, T, V, W or Y, and / or, the 369th amino acid residue is replaced by E, P, S or T, and / or, the 370th amino acid residue is replaced by R or V, and / or, the 371st amino acid residue is replaced by K or W, and / or, the 372nd amino acid residue is replaced by D or F, and / or, the 373rd amino acid residue is replaced by A, F, P or Y, and / or, the 375th amino acid residue is replaced by E or Q, and / or, the 376th amino acid residue is replaced by K, R or T, and / or, the 377th amino acid residue is replaced by R, and / or, the 379th amino acid residue is replaced by G, and / or,The 380th amino acid residue is replaced with E, P, or W, and / or, the 382nd amino acid residue is replaced with C, and / or, the 383rd amino acid residue is replaced with Q, R, T, or V, and / or, the 384th amino acid residue is replaced with A, C, or Y, and / or, the 386th amino acid residue is replaced with R, T, or W, and / or, the 388th amino acid residue is replaced with N, P, or S, and / or, the 389th amino acid residue is replaced with T, and / or, the 390th amino acid residue is replaced with L or Y, and / or, the 393rd amino acid residue is replaced with L, P, T, or W, and / or, the 395th amino acid residue is replaced with E or K, and / or, the 396th amino acid residue is replaced with E, K, L, N, R, S, or T.,

[0014] In one embodiment, the mutant has the 21st amino acid residue in the amino acid sequence shown in SEQ ID NO:1 replaced by K, and / or the 35th amino acid residue replaced by M, and / or the 41st amino acid residue replaced by Q, and / or the 42nd amino acid residue replaced by I, and / or the 44th amino acid residue replaced by Y, and / or the 55th amino acid residue replaced by T, and / or the 56th amino acid residue replaced by R or T, and / or the 64th amino acid residue replaced by I, and / or the 73rd amino acid residue replaced by A, and / or the 75th amino acid residue replaced by L, and / or the 83rd amino acid residue replaced by A, and / or the 87th amino acid residue replaced by W, and / or the 92nd amino acid residue replaced by P, and / or the 97th amino acid residue replaced by R, and / or the 103rd amino acid residue replaced by A, and / or the 114th amino acid residue replaced by A, and / or the 119th amino acid residue replaced by W, and / or the 123rd amino acid residue replaced by P, and / or the 148th amino acid residue replaced by F, and / or the 150th amino acid residue replaced by K, and / or the 155th amino acid residue replaced by I, and / or the 158th amino acid residue replaced by C, and / or the 170th amino acid residue replaced by N, and / or the 179th amino acid residue replaced by T, and / or the 190th amino acid residue replaced by T, and / or the 199th amino acid residue replaced by F, and / or the 201st amino acid residue replaced by M, and / or the 213th amino acid residue replaced by V, and / or the 219th amino acid residue replaced by P, and / or the 221st amino acid residue replaced by W, and / or the 222nd amino acid residue replaced by K, and / or the 229th amino acid residue replaced by R, and / or the 242nd amino acid residue replaced by W, and / or the 249th amino acid residue replaced by W, and / or the 253rd amino acid residue replaced by A, and / or the 257th amino acid residue replaced by R or V, and / or the 261st amino acid residue replaced by D, and / or the 284th amino acid residue replaced by R, and / or the 287th amino acid residue replaced by M, and / or the 297th amino acid residue replaced by T, and / or the 299th amino acid residue replaced by N, and / or the 304th amino acid residue replaced by F, and / or the 315th amino acid residue replaced by P, and / or the 318th amino acid residue replaced by R, and / or the 344th amino acid residue replaced by R, and / or the 356th amino acid residue replaced by I, and / or the 370th amino acid residue replaced by R, and / or the 373rd amino acid residue replaced by A, and / or the 376th amino acid residue replaced by T, and / or the 377th amino acid residue replaced by R, and / or the 379th amino acid residue replaced by G, and / orThe 388th amino acid residue is replaced by N, and / or the 393rd amino acid residue is replaced by L, and / or the 395th amino acid residue is replaced by K.

[0015] In one embodiment, the mutant has differences in amino acid residues at 2 to 14 positions in the amino acid sequence as shown in SEQ ID NO:1.

[0016] In one embodiment, the mutant has differences in amino acid residues at least at the following positions in the amino acid sequence as shown in SEQ ID NO:1: the 297th and 315th positions; preferably, the 297th amino acid residue is replaced by T, and the 315th amino acid residue is replaced by P.

[0017] In one embodiment, the mutant has differences in amino acid residues at least at the following positions in the amino acid sequence as shown in SEQ ID NO:1: the 297th, 315th, 373rd and 376th positions; preferably, the 297th amino acid residue is replaced by T, the 315th amino acid residue is replaced by P, the 373rd amino acid residue is replaced by A, and the 376th amino acid residue is replaced by T.

[0018] In one embodiment, the mutant has differences in amino acid residues at least at the following positions in the amino acid sequence as shown in SEQ ID NO:1: the 44th, 75th, 297th and 376th positions; preferably, the 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 297th amino acid residue is replaced by T, and the 276th amino acid residue is replaced by T.

[0019] In one embodiment, the mutant has differences in amino acid residues at least at the following positions in the amino acid sequence as shown in SEQ ID NO:1: the 44th, 75th, 297th and 376th positions; preferably, the 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 297th amino acid residue is replaced by T, and the 276th amino acid residue is replaced by T.

[0020] In one embodiment, the mutant has at least the following differences in amino acid residues at the following positions on the amino acid sequence shown in SEQ ID NO: 1: positions 35, 44, 75, 92, 150, 297, 315, 373, 376, and 393; preferably, the amino acid residue at position 35 is replaced by M, the amino acid residue at position 44 is replaced by Y, the amino acid residue at position 75 is replaced by L, the amino acid residue at position 92 is replaced by P, the amino acid residue at position 150 is replaced by K, the amino acid residue at position 297 is replaced by T, the amino acid residue at position 315 is replaced by P, the amino acid residue at position 373 is replaced by A, the amino acid residue at position 376 is replaced by T, and the amino acid residue at position 393 is replaced by L.

[0021] In one embodiment, the mutant has at least the following differences in amino acid residues at the following positions on the amino acid sequence shown in SEQ ID NO: 1: positions 44, 56, 75, 92, 150, 257, 297, 315, 373, and 376; preferably, the amino acid residue at position 44 is replaced by Y, the amino acid residue at position 56 is replaced by R or T, the amino acid residue at position 75 is replaced by L, the amino acid residue at position 92 is replaced by P, the amino acid residue at position 150 is replaced by K, the amino acid residue at position 257 is replaced by V or R, the amino acid residue at position 297 is replaced by T, the amino acid residue at position 315 is replaced by P, the amino acid residue at position 373 is replaced by A, and the amino acid residue at position 376 is replaced by T.

[0022] In one embodiment, the differences of the mutant on the amino acid sequence shown in SEQ ID NO: 1 are shown in the following table:

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031] In one embodiment, the compound shown in Formula II is The corresponding compound shown in Formula I is

[0032] In one embodiment, the mass ratio of the enol reductase or its mutant to the compound shown in Formula II is (0.1 - 20):1, such as (0.25 - 4):1, and further such as 0.25:1, 0.4:1, 0.5:1, 1:1 or 2:1.

[0033] In one embodiment, the solvent is a buffer solution, such as one or more selected from phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS) and triethanolamine buffer (TEOA). The pH of the buffer solution can be 5.5 - 9.5, such as 6.5, 7.0, 7.5 or 8.5.

[0034] In one embodiment, the solvent is phosphate buffer (PBS) with a pH of 6.5, phosphate buffer (PBS) with a pH of 7.0, phosphate buffer (PBS) with a pH of 7.5, phosphate buffer (PBS) with a pH of 8.5, triethanolamine buffer (TEOA) with a pH of 6.5 or tris(hydroxymethyl)aminomethane buffer (TRIS) with a pH of 6.5.

[0035] In one embodiment, the volume - mass ratio of the solvent to the compound shown in Formula II is 10 - 2000 mL / g, such as 10 - 200 mL / g, and further such as 14 mL / g, 35 mL / g or 200 mL / g.

[0036] In one embodiment, the reduction reaction further comprises a co - solvent, which is used to improve the solubility of the compound shown in Formula II. It can be a conventional co - solvent in the art, such as DMSO. Preferably, the volume - mass ratio of the co - solvent to the compound shown in Formula II is 10 - 500 mL / g, such as 10 - 30 mL / g, and further such as 10 mL / g, 15 mL / g or 30 mL / g.

[0037] In one embodiment, the coenzyme is a reduced coenzyme, such as nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), and preferably nicotinamide adenine dinucleotide phosphate (NADPH).

[0038] In one embodiment, in the reduction reaction, when the coenzyme is an oxidized coenzyme or its salt, or when the amount of the reduced coenzyme is not sufficient, the reduction reaction further comprises a coenzyme regeneration system. The coenzyme regeneration system includes a reductase or dehydrogenase, and a hydrogen donor to recycle the coenzyme.

[0039] Preferably, the coenzyme regeneration system is any one of the following systems:

[0040] System 1: The carbonyl reductase (KRED) is preferably a ketoreductase or an alcohol dehydrogenase (ADH), and a hydrogen donor such as isopropanol;

[0041] System 2: Glucose dehydrogenase (GDH), and a hydrogen donor such as glucose.

[0042] In a certain embodiment, in the reduction reaction, the coenzyme is an oxidized coenzyme or its salt. At this time, the reduction reaction includes a coenzyme regeneration system, and the coenzyme regeneration system includes a reductase or a dehydrogenase, and a hydrogen donor.

[0043] Preferably, the oxidized coenzyme or its salt is nicotinamide adenine dinucleotide (NAD+), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide disodium salt (NAD-Na2) or nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2), preferably oxidized nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2), such as nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2).

[0044] Preferably, the mass ratio of the reductase or dehydrogenase to the compound shown in Formula II is (0.05 - 6):1, such as 0.05:1, 0.1:1 or 0.2:1.

[0045] Preferably, the mass ratio of the reductase or dehydrogenase to the hydrogen donor is (0.05 - 0.2):1, such as 0.05:1, 0.1:1 or 0.2:1.

[0046] Preferably, the mass ratio of the oxidized coenzyme to the compound shown in Formula II is (0.001 - 0.5):1, such as (0.01 - 0.1):1, and further such as 0.05:1.

[0047] Preferably, the coenzyme regeneration system includes a dehydrogenase and a hydrogen donor. The dehydrogenase is glucose dehydrogenase, and the hydrogen donor is glucose, such as D-glucose.

[0048] In a certain embodiment, the preparation method of the compound shown in Formula I includes the following steps: In the solvent, in the presence of the alkene reductase or its mutant, the dehydrogenase or reductase, the oxidized coenzyme and the hydrogen donor, the compound shown in Formula II undergoes the reduction reaction as described above to prepare the compound shown in Formula I.

[0049] In one embodiment, the reaction temperature of the reduction reaction is 0 to 37 °C, such as 4 °C, 7 °C, 17 °C, 18 °C, 20 °C or 27 °C.

[0050] The reduction reaction may take the disappearance or no longer reaction of the compound shown in Formula II as the reaction end point.

[0051] In one embodiment, the reaction time of the reduction reaction is 0.5 to 42 h, such as 4 to 24 h, such as 8 h, 16 h or 24 h.

[0052] The present invention also provides a mutant of an eno reductase, which is the mutant defined in any of the above embodiments.

[0053] In one embodiment of the present invention, the mutant is the mutant 002 to mutant 213 as described above.

[0054] The present invention also provides an isolated nucleic acid, wherein the nucleic acid encodes the mutant of the eno reductase as described above.

[0055] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid as described above.

[0056] The present invention also provides a transformant, wherein the transformant contains the recombinant expression vector as described above.

[0057] The present invention also provides a method for preparing a mutant of an eno reductase, which comprises the following step: culturing the transformant as described above and obtaining a culture containing the mutant of the eno reductase.

[0058] The present invention also provides an application of the above eno reductase or its mutant in the asymmetric reduction of prochiral olefins. Preferably, the prochiral olefin is the compound shown in Formula II as described in any of the above embodiments.

[0059] In one embodiment of the application, the reaction operation and conditions of the asymmetric reduction are as described in the reduction reaction of any of the above embodiments.

[0060] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0061] The reagents and raw materials used in the present invention are all commercially available.

[0062] The amino acid abbreviation symbols in the present invention are all conventional in the art without special instructions, and the amino acids corresponding to the specific abbreviation symbols are shown in the following table.

[0063]

[0064] The positive and progressive effects of the present invention are as follows: The asymmetric reduction reaction of olefins catalyzed by the eno reductase in the present invention has one or more of the following advantages: high raw material conversion rate, high yield, high chiral purity of the product, high selectivity, and mild conditions. Detailed implementation manners

[0065] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications. The eno reductase of the present invention can be expressed and purified by recombinant expression technology or prepared by total chemical synthesis, and these methods are all conventional means for obtaining enzyme proteins well-known in the art.

[0066] In the following examples, Compound 2 is Compound 1 is

[0067] The sequences of the eno reductase mutants involved in the following examples are shown in Table 1 and can be obtained by total chemical synthesis or by computer-aided design and high-throughput screening methods. The computer-aided design and high-throughput screening methods are all conventional means for constructing mutants in the art.

[0068] The compound structure and purity test methods in the following examples are as follows:

[0069] The test method uses supercritical fluid chromatography SFC (Waters, Supercritical Fluid Chromatography), the chromatographic column is CHIRALPAK IG-3, 150×4.6 mm, inner diameter 3 μm, PN: 87524; the mobile phases are CO2 (mobile phase A) and 0.2% MIPA in MeOH (mobile phase B) respectively; isocratic elution is carried out with a mixed phase of 82% mobile phase A and 18% mobile phase B (where 82% and 18% are volume fractions); the flow rate is 2.5 mL / min; the running time is 8.0 min; the detection wavelength is 294 nm, the peak time of Compound 2 is 6.12 min, the peak time of Compound 1(R) is 5.35 min (the same as the retention time of the Compound 1 standard), and the peak time of the isomer (S) of Compound 1 is 4.73 min.

[0070] Sample yield: {Compound 1(S) + Compound 1(R)} / Compound 2

[0071] e.e. value: {Compound 1(R) - Compound 1(S)} / {(Compound 1(R) + Compound 1(S)}

[0072] Example 1: In PBS buffer, the eno reductase catalyzes Compound 2 to Compound 1.

[0073] Add 0.4 g of eno reductase mutant 058, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of Compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of 2-methyltetrahydrofuran (CAS 96-47-9), take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) to finally obtain 0.870 g of the product, with a yield of 87% and an ee value of 66%.

[0074] Example 2: In TEOA buffer, eno reductase catalyzes Compound 2 to Compound 1.

[0075] Add 0.4 g of eno reductase mutant 031, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of TEOA (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of Compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) to finally obtain 0.712 g of the product, with a yield of 71.2% and an ee value of 69%.

[0076] Example 3: In Tris buffer, eno reductase catalyzes Compound 2 to Compound 1.

[0077] Add 0.4 g of eno reductase mutant 194, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of Tris (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of Compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) to finally obtain 0.731 g of the product, with a yield of 73.1% and an ee value of 70%.

[0078] Example 4: The ene reductase catalyzes compound 2 to compound 1 under the condition of pH = 7.

[0079] Add 0.4 g of ene reductase mutant 073, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 7) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove moisture, then filter to obtain the filtrate, and distill it under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.90 g of the product is obtained, with a yield of 90% and an ee value of 98%.

[0080] Example 5: The ene reductase catalyzes compound 2 to compound 1 under the condition of pH = 7.5.

[0081] Add 0.4 g of ene reductase mutant 088, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 7.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove moisture, then filter to obtain the filtrate, and distill it under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.774 g of the product is obtained, with a yield of 77.4% and an ee value of 94.0%.

[0082] Example 6: The ene reductase catalyzes compound 2 to compound 1 under the condition of pH = 8.5.

[0083] Add 0.4 g of ene reductase mutant 148, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 8.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove moisture, then filter to obtain the filtrate, and distill it under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.718 g of the product is obtained, with a yield of 72% and an ee value of 75%.

[0084] Example 7: Under the condition that the volume ratio of the co-solvent DMSO to compound 2 is 10, the ene reductase catalyzes compound 2 to compound 1.

[0085] Add 0.4 g of ene reductase mutant 206, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React 1 g of compound 2 (dissolved in 10 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.892 g of the product is obtained, with a yield of 89.2% and an ee value of 63%.

[0086] Example 8: Under the condition that the volume ratio of the co-solvent DMSO to compound 2 is 30, the ene reductase catalyzes compound 2 to compound 1.

[0087] Add 0.4 g of ene reductase mutant 099, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 100 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React 1 g of compound 2 (dissolved in 30 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.6980 g of the product is obtained, with a yield of 69.8% and an ee value of 79%.

[0088] Example 9: Under the condition that the mass ratio of the ene reductase to compound 2 is 0.5:1, the ene reductase catalyzes compound 2 to compound 1.

[0089] Add 0.5 g of eno reductase mutant 162, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase and add anhydrous sodium sulfate to remove moisture, then filter to obtain a filtrate, and carry out vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.883 g of the product is obtained, with a yield of 88.3% and an ee value of >71.2%.

[0090] Example 10: Under the condition that the mass ratio of eno reductase to compound 2 is 1:1, eno reductase catalyzes compound 2 to compound 1.

[0091] Add 1 g of eno reductase mutant 184, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase and add anhydrous sodium sulfate to remove moisture, then filter to obtain a filtrate, and carry out vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.918 g of the product is obtained, with a yield of 91.8% and an ee value of 94%.

[0092] Example 11: Under the condition that the mass ratio of eno reductase to compound 2 is 2:1, eno reductase catalyzes compound 2 to compound 1.

[0093] Add 2 g of eno reductase mutant 095, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase and add anhydrous sodium sulfate to remove moisture, then filter to obtain a filtrate, and carry out vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.922 g of the product is obtained, with a yield of 92.2% and an ee value of 95%.

[0094] Example 12: Under the condition that the mass ratio of glucose dehydrogenase to Compound 2 is 0.05:1, ene reductase catalyzes Compound 2 into Compound 1.

[0095] Add 0.4 g of ene reductase mutant 092, 0.05 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose, and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of Compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and perform vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.913 g of the product is obtained, with a yield of 91.3% and an ee value of 93%.

[0096] Example 13: Under the condition that the mass ratio of glucose dehydrogenase to Compound 2 is 0.1:1, ene reductase catalyzes Compound 2 into Compound 1.

[0097] Add 0.4 g of ene reductase mutant 216, 0.1 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose, and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of Compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and perform vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.729 g of the product is obtained, with a yield of 72.9% and an ee value of 85.1%.

[0098] Example 14: Under the condition that the mass ratio of β-nicotinamide adenine dinucleotide disodium phosphate to Compound 2 is 0.01:1, ene reductase catalyzes Compound 2 into Compound 1.

[0099] Add 0.4 g of eno reductase mutant 032, 0.2 g of glucose dehydrogenase, 0.01 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction and add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.907 g of the product is obtained, with a yield of 90.7% and an ee value of 66.2%.

[0100] Example 15: Under the condition that the mass-volume ratio of compound 2 to the buffer solution is 1:14 g / mL, eno reductase catalyzes compound 2 to compound 1.

[0101] Add 0.4 g of eno reductase mutant 142, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 14 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction and add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.821 g of the product is obtained, with a yield of 82.1% and an ee value of 69.2%.

[0102] Example 16: Under the condition that the mass-volume ratio of compound 2 to the buffer solution is 1:200 g / mL, eno reductase catalyzes compound 2 to compound 1.

[0103] Add 0.25 g of eno reductase mutant 188, 0.2 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 200 mL of PBS (pH = 6.5) buffer solution to a 250 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. React with 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction and add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and distill under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.753 g of the product is obtained, with a yield of 75.3% and an ee value of 79.8%.

[0104] Example 17: At a reaction temperature of 4 °C, eno reductase catalyzes compound 2 to compound 1.

[0105] Add 0.4 g of eno reductase mutant 116, 0.2 g of glucose dehydrogenase, 0.05 g of β - nicotinamide adenine dinucleotide disodium phosphate, 1 g of D - glucose and 35 mL of PBS (pH = 6.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 4 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain the filtrate, and perform vacuum distillation at a vacuum degree of - 0.09 MPa (gauge pressure). Finally, 0.702 g of the product is obtained, with a yield of 70.2% and an ee value of 68.7%.

[0106] Example 18: At a reaction temperature of 17 °C, eno reductase catalyzes compound 2 to compound 1.

[0107] Add 0.4 g of eno reductase mutant 090, 0.2 g of glucose dehydrogenase, 0.05 g of β - nicotinamide adenine dinucleotide disodium phosphate, 1 g of D - glucose and 35 mL of PBS (pH = 6.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 17 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain the filtrate, and perform vacuum distillation at a vacuum degree of - 0.09 MPa (gauge pressure). Finally, 0.850 g of the product is obtained, with a yield of 85% and an ee value of 78.1%.

[0108] Example 19: At a reaction temperature of 27 °C, eno reductase catalyzes compound 2 to compound 1.

[0109] Add 0.4 g of eno reductase mutant 162, 0.2 g of glucose dehydrogenase, 0.05 g of β - nicotinamide adenine dinucleotide disodium phosphate, 1 g of D - glucose and 35 mL of PBS (pH = 6.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 27 °C and react for 8 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain the filtrate, and carry out vacuum distillation under a vacuum degree of - 0.09 MPa (gauge pressure). Finally, 0.882 g of the product is obtained, with a yield of 88.2% and an ee value of 88%.

[0110] Example 20: At a reaction time of 4 h, eno reductase catalyzes compound 2 to compound 1.

[0111] Add 0.4 g of eno reductase mutant 25, 0.2 g of glucose dehydrogenase, 0.05 g of β - nicotinamide adenine dinucleotide disodium phosphate, 1 g of D - glucose and 35 mL of PBS (pH = 6.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 4 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain the filtrate, and carry out vacuum distillation under a vacuum degree of - 0.09 MPa (gauge pressure). Finally, 0.729 g of the product is obtained, with a yield of 72.9% and an ee value of 84.1%.

[0112] Example 21: At a reaction time of 24 h, eno reductase catalyzes compound 2 to compound 1.

[0113] Add 0.4 g of eno reductase mutant 020, 0.2 g of glucose dehydrogenase, 0.05 g of β - nicotinamide adenine dinucleotide disodium phosphate, 1 g of D - glucose and 35 mL of PBS (pH = 6.5) buffer solution into a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 24 h. Centrifuge the reaction solution, extract the lower precipitate with 50.0 mL of dimethyltetrahydrofuran. After extraction, take out the organic phase, add anhydrous sodium sulfate to remove water, then filter to obtain the filtrate, and carry out vacuum distillation under a vacuum degree of - 0.09 MPa (gauge pressure). Finally, 0.901 g of the product is obtained, with a yield of 90% and an ee value of 92%.

[0114] In Example 22, the ene-reductase catalyzed compound 2 to compound 1 under the condition that the mass ratio of glucose dehydrogenase to compound 2 was 0.05:1.

[0115] Add 0.4 g of ene-reductase mutant 214, 0.05 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and perform vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.732 g of the product is obtained, the yield is 73%, and the ee value is 30%.

[0116] In Example 23, the ene-reductase catalyzed compound 2 to compound 1 under the condition that the mass ratio of glucose dehydrogenase to compound 2 was 0.05:1.

[0117] Add 0.4 g of ene-reductase mutant 215, 0.05 g of glucose dehydrogenase, 0.05 g of β-nicotinamide adenine dinucleotide disodium phosphate, 1 g of D-glucose and 35 mL of PBS (pH = 6.5) buffer solution to a 50 mL jacketed flask, and stir magnetically until all the enzymes are dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 15 mL of DMSO) at 7 °C and react for 8 h. Centrifuge the reaction solution, extract the lower layer precipitate with 50.0 mL of dimethyltetrahydrofuran, take out the organic phase after extraction, add anhydrous sodium sulfate to remove water, then filter to obtain a filtrate, and perform vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.588 g of the product is obtained, the yield is 59%, and the ee value is 72%.

[0118] Example 24

[0119] The reaction conditions were as shown in Example 1, except that the above-mentioned ene-reductase mutant 058 was replaced with mutants 002 - 216 and wild-type ene-reductase. Mutants 002 - 216 were obtained by mutating the wild-type ene-reductase. The results are shown in Table 1 below:

[0120] Table 1 Performance parameters of ene-reductase and its mutants

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] The above embodiments include the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a compound represented by formula I, which comprises the following steps: in a solvent, in the presence of an enoyl reductase or a mutant thereof, and a coenzyme, the compound represented by formula II undergoes the following reduction reaction to prepare the compound represented by formula I, that's all. Among them, R 1 is methyl, ethyl, n-propyl, isopropyl or hydroxyl; R 2 is methyl, ethyl, n-propyl, aldehyde group, hydroxyl or carboxyl group; The coenzyme is a coenzyme of the enoyl reductase or its mutant. The amino acid sequence of the enoyl reductase is as shown in SEQ ID NO:

1. The mutant has differences in amino acid residues at one or more sites selected from the following positions in the amino acid sequence shown in SEQ ID NO: 1: position 2, position 3, position 4, position 5, position 8, position 9, position 10, position 12, position 13, position 17, position 19, position 20, position 21, position 23, position 24, position 26, position 27, position 28, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 46, position 51, position 53, position 55, position 56, position 57, position 58, position 59, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 71, position 73, position 74, position 75, position 76, position 77, position 80, position 81, position 82, position 83, position 85, position 86, position 87, position 88, position 89, position 92, position 95, position 96, position 97, position 98, position 99, position 100, position 101, position 102, position 103, position 104, position 105, position 106, position 113, position 114, position 115, position 116, position 117, position 118, position 119, position 120, position 121, position 122, position 123, position 124, position 125, position 126, position 128, position 135, position 136, position 137, position 145, position 148, position 149, position 150, position 151, position 152, position 153, position 154, position 155, position 156, position 157, position 158, position 159, position 160, position 161, position 162, position 167, position 168, position 169, position 170, position 172, position 173, position 174, position 175, position 177, position 179, position 180, position 181, position 184, position 189, position 190, position 191, position 192, position 193, position 194, position 195, position 196, position 197, position 198, position 199, position 200, position 201, position 207, position 208, position 209, position 210, position 211, position 212, position 213, position 214, position 215, position 216, position 217, position 218, position 219, position 221, position 222, position 223, position 224, position 225, position 226, position 227, position 228, position 229, position 231, position 232, position 233, position 234, position 242, position 243, position 244, position 245, position 246, position 247, position 248The 249th, 250th, 251st, 252nd, 253rd, 254th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 268th, 269th, 270th, 271st, 272nd, 273rd, 274th, 277th, 278th, 283rd, 284th, 285th, 287th, 288th, 289th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th, 297th, 298th, 299th, 300th, 301st, 302nd, 303rd, 304th, 305th, 309th, 310th, 311th, 312th, 313th, 314th, 315th, 316th, 317th, 318th, 320th, 321st, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 333rd, 334th, 335th, 336th, 337th, 338th, 339th, 340th, 341st, 342nd, 343rd, 344th, 346th, 349th, 350th, 351st, 352nd, 353rd, 354th, 356th, 357th, 361st, 362nd, 363rd, 367th, 368th, 369th, 370th, 371st, 372nd, 373rd, 375th, 376th, 377th, 379th, 380th, 382nd, 383rd, 384th, 386th, 388th, 389th, 390th, 393rd, 395th and 396th.

2. The preparation method of the compound shown in Formula I as described in Claim 1, characterized in that, The difference is a deletion, an addition or a substitution, preferably a substitution. Preferably, the mutant has differences in amino acid residues at 2 to 14 positions on the amino acid sequence shown in SEQ ID NO:

1.

3. The preparation method of the compound shown in Formula I as described in Claim 1, characterized in that, The mutant is one in which the amino acid residue at the 2nd position of the amino acid sequence shown in SEQ ID NO: 1 is replaced with K, N or V, and / or the amino acid residue at the 3rd position is replaced with G, I or K, and / or the amino acid residue at the 4th position is replaced with A, D or E, and / or the amino acid residue at the 5th position is replaced with E or G, and / or the amino acid residue at the 8th position is replaced with A or K, and / or the amino acid residue at the 9th position is replaced with D, and / or the amino acid residue at the 10th position is replaced with M, N or V, and / or, and / or the amino acid residue at the 12th position is replaced with M, Q, R or T, and / or the amino acid residue at the 13th position is replaced with D or V, and / or the amino acid residue at the 17th position is replaced with C, N or Y, and / or the amino acid residue at the 19th position is replaced with A, and / or the amino acid residue at the 20th position is replaced with D, and / or the amino acid residue at the 21st position is replaced with E, F or K, and / or the amino acid residue at the 23rd position is replaced with W, and / or the amino acid residue at the 24th position is replaced with D, R or T, and / or the amino acid residue at the 26th position is replaced with Q or W, and / or the amino acid residue at the 27th position is replaced with H or T, and / or the amino acid residue at the 28th position is replaced with G or W, and / or the amino acid residue at the 31st position is replaced with I or W, and / or the amino acid residue at the 32nd position is replaced with N or Y, and / or the amino acid residue at the 33rd position is replaced with H or T, and / or the amino acid residue at the 34th position is replaced with C, L, V or W, and / or the amino acid residue at the 35th position is replaced with F, M, T or Y, and / or the amino acid residue at the 36th position is replaced with M, and / or the amino acid residue at the 37th position is replaced with G, and / or the amino acid residue at the 38th position is replaced with D, and / or the amino acid residue at the 39th position is replaced with E, G, I, P or T, and / or the amino acid residue at the 40th position is replaced with A or D, and / or the amino acid residue at the 41st position is replaced with A, D, L or Q, and / or the amino acid residue at the 42nd position is replaced with G or I, and / or the amino acid residue at the 43rd position is replaced with C, E, G, P or V, and / or the amino acid residue at the 44th position is replaced with W or Y, and / or the amino acid residue at the 46th position is replaced with E or I, and / or the amino acid residue at the 51st position is replaced with N, and / or the amino acid residue at the 53rd position is replaced with E, G or I, and / or the amino acid residue at the 55th position is replaced with C, G, R or T, and / or the amino acid residue at the 56th position is replaced with I, K, R or T, and / or the amino acid residue at the 57th position is replaced with I, K, M or W, and / or the amino acid residue at the 58th position is replaced with G or N, and / or the amino acid residue at the 59th position is replaced with F, M or V, and / or the amino acid residue at the 61st position is replaced with C or Y, and / or the amino acid residue at the 62nd position is replaced with M or V, and / or the amino acid residue at the 63rd position is replaced with Y, and / or the amino acid residue at the 64th position is replaced with D or I, and / or the amino acid residue at the 65th position is replaced with C, D or W, and / or the amino acid residue at the 66th position is replaced with L, and / orThe 67th amino acid residue is replaced with L or N, and / or, the 68th amino acid residue is replaced with P or Q, and / or, the 69th amino acid residue is replaced with E, and / or, the 71st amino acid residue is replaced with G or V, and / or, the 73rd amino acid residue is replaced with A, Q or V, and / or, the 74th amino acid residue is replaced with W, and / or, the 75th amino acid residue is replaced with C, E, L or S, and / or, the 76th amino acid residue is replaced with N or V, and / or, the 77th amino acid residue is replaced with R, V or Y, and / or, the 80th amino acid residue is replaced with K or N, and / or, the 81st amino acid residue is replaced with C, I or T, and / or, the 82nd amino acid residue is replaced with D or T, and / or, the 83rd amino acid residue is replaced with A, F, H, I or M, and / or, the 85th amino acid residue is replaced with G or L, and / or, the 86th amino acid residue is replaced with R or S, and / or, the 87th amino acid residue is replaced with R or W, and / or, the 88th amino acid residue is replaced with C, F, K or R, and / or, the 89th amino acid residue is replaced with G, T or W, and / or, the 92nd amino acid residue is replaced with H, P or W, and / or, the 95th amino acid residue is replaced with L or R, and / or, the 96th amino acid residue is replaced with E, and / or, the 97th amino acid residue is replaced with P or R, and / or, the 98th amino acid residue is replaced with A, I, K, M, N or Y, and / or, the 99th amino acid residue is replaced with I, N or R, and / or, the 100th amino acid residue is replaced with D, E or K, and / or, the 101st amino acid residue is replaced with F or H, and / or, the 102nd amino acid residue is replaced with P, T or V, and / or, the 103rd amino acid residue is replaced with A or E, and / or, the 104th amino acid residue is replaced with L or S, and / or, the 105th amino acid residue is replaced with E, and / or, the 106th amino acid residue is replaced with I, K or W, and / or, the 113th amino acid residue is replaced with A, E, I or Q, and / or, the 114th amino acid residue is replaced with Q, R or T, and / or, the 115th amino acid residue is replaced with M or R, and / or, the 116th amino acid residue is replaced with H or W, and / or, the 117th amino acid residue is replaced with N, and / or, the 118th amino acid residue is replaced with E, and / or, the 119th amino acid residue is replaced with E or Y, and / or, the 120th amino acid residue is replaced with I, and / or, the 121st amino acid residue is replaced with D, F, K or N, and / or, the 122nd amino acid residue is replaced with R, and / or, the 123rd amino acid residue is replaced with E, F, K or P, and / or, the 124th amino acid residue is replaced with H, I or T, and / or, the 125th amino acid residue is replaced with M, Q or S, and / or, the 126th amino acid residue is replaced with A, G, I or L, and / or, the 128th amino acid residue is replaced with W, and / or, the 135th amino acid residue is replaced with Vand / or, the 136th amino acid residue is replaced with A or G, and / or, the 137th amino acid residue is replaced with K, and / or, the 145th amino acid residue is replaced with L, and / or, the 148th amino acid residue is replaced with A, F, H or V, and / or, the 149th amino acid residue is replaced with F, and / or, the 150th amino acid residue is replaced with F, K, N or P, and / or, the 151st amino acid residue is replaced with N, and / or, the 152nd amino acid residue is replaced with E, and / or, the 153rd amino acid residue is replaced with D, N or S, and / or, the 154th amino acid residue is replaced with L or T, and / or, the 155th amino acid residue is replaced with I or Y, and / or, the 156th amino acid residue is replaced with F, and / or, the 157th amino acid residue is replaced with L or W, and / or, the 158th amino acid residue is replaced with C, Q, R or T, and / or, the 159th amino acid residue is replaced with H, I or P, and / or, the 160th amino acid residue is replaced with L, and / or, the 161st amino acid residue is replaced with W, and / or, the 162nd amino acid residue is replaced with K or W, and / or, the 167th amino acid residue is replaced with E, P or S, and / or, the 168th amino acid residue is replaced with F, H, I or Y, and / or, the 169th amino acid residue is replaced with E, I or T, and / or, the 170th amino acid residue is replaced with C, H, K, M, N or P, and / or, the 172nd amino acid residue is replaced with L, and / or, the 173rd amino acid residue is replaced with I, and / or, the 174th amino acid residue is replaced with E, H or R, and / or, the 175th amino acid residue is replaced with W, and / or, the 177th amino acid residue is replaced with F, S or W, and / or, the 179th amino acid residue is replaced with F, G, P, T or V, and / or, the 180th amino acid residue is replaced with I or M, and / or, the 181st amino acid residue is replaced with F or Y, and / or, the 184th amino acid residue is replaced with F or P, and / or, the 189th amino acid residue is replaced with T or Y, and / or, the 190th amino acid residue is replaced with D, N or T, and / or, the 191st amino acid residue is replaced with F, K, L or R, and / or, the 192nd amino acid residue is replaced with E, G, I, M, R or S, and / or, the 193rd amino acid residue is replaced with F, L, M or T, and / or, the 194th amino acid residue is replaced with C, D, P or T, and / or, the 195th amino acid residue is replaced with E, H, K or Y, and / or, the 196th amino acid residue is replaced with R, and / or, the 197th amino acid residue is replaced with F or S, and / or, the 198th amino acid residue is replaced with Q, and / or, the 199th amino acid residue is replaced with F, and / or, the 200th amino acid residue is replaced with R, and / or, the 201st amino acid residue is replaced with M, and / or, the 207th amino acid residue is replaced with E, P or Q, and / or,The 208th amino acid residue is replaced with D, M, R, or V, and / or, the 209th amino acid residue is replaced with M, and / or, the 210th amino acid residue is replaced with D, H, or I, and / or, the 211th amino acid residue is replaced with A or R, and / or, the 212th amino acid residue is replaced with A, and / or, the 213th amino acid residue is replaced with H, V, or W, and / or, the 214th amino acid residue is replaced with M or N, and / or, the 215th amino acid residue is replaced with W or Y, and / or, the 216th amino acid residue is replaced with C or N, and / or, the 217th amino acid residue is replaced with M, R, or W, and / or, the 218th amino acid residue is replaced with C, F, L, P, or Q, and / or, the 219th amino acid residue is replaced with D or P, and / or, the 221st amino acid residue is replaced with C, E, K, W, or Y, and / or, the 222nd amino acid residue is replaced with K, R, V, or Y, and / or, the 223rd amino acid residue is replaced with H or L, and / or, the 224th amino acid residue is replaced with G, and / or, the 225th amino acid residue is replaced with A, and / or, the 226th amino acid residue is replaced with N, and / or, the 227th amino acid residue is replaced with D, and / or, the 228th amino acid residue is replaced with D, N, or R, and / or, the 229th amino acid residue is replaced with C, N, R, W, or Y, and / or, the 231st amino acid residue is replaced with K, Q, or S, and / or, the 232nd amino acid residue is replaced with A, D, G, N, or P, and / or, the 233rd amino acid residue is replaced with D, and / or, the 234th amino acid residue is replaced with H, I, M, or P, and / or, the 242nd amino acid residue is replaced with A, E, F, S, or W, and / or, the 243rd amino acid residue is replaced with A, F, H, or T, and / or, the 244th amino acid residue is replaced with Q, and / or, the 245th amino acid residue is replaced with A or V, and / or, the 246th amino acid residue is replaced with F, H, K, or Q, and / or, the 247th amino acid residue is replaced with C, W, or Y, and / or, the 248th amino acid residue is replaced with L, and / or, the 249th amino acid residue is replaced with W, and / or, the 250th amino acid residue is replaced with E, H, or Q, and / or, the 251st amino acid residue is replaced with A, D, R, or S, and / or, the 252nd amino acid residue is replaced with V, and / or, the 253rd amino acid residue is replaced with A, C, T, or Y, and / or, the 254th amino acid residue is replaced with G or V, and / or, the 256th amino acid residue is replaced with A, E, H, or V, and / or, the 257th amino acid residue is replaced with L, R, T, or V, and / or, the 258th amino acid residue is replaced with C or Q, and / or, the 259th amino acid residue is replaced with G, and / or, the 260th amino acid residue is replaced with D or Q, and / or, the 261st amino acid residue is replaced with D or Q, and / or, the 262nd amino acid residue is replaced with D, K, or Rand / or, the 263rd amino acid residue is replaced with C, and / or, the 264th amino acid residue is replaced with I, and / or, the 268th amino acid residue is replaced with I, M, P or S, and / or, the 269th amino acid residue is replaced with G or S, and / or, the 270th amino acid residue is replaced with K, and / or, the 271st amino acid residue is replaced with F or K, and / or, the 272nd amino acid residue is replaced with A, D or R, and / or, the 273rd amino acid residue is replaced with G, K, M or W, and / or, the 274th amino acid residue is replaced with G, M or P, and / or, the 277th amino acid residue is replaced with L, and / or, the 278th amino acid residue is replaced with C, and / or, the 283rd amino acid residue is replaced with E or F, and / or, the 284th amino acid residue is replaced with D, K or R, and / or, the 285th amino acid residue is replaced with H, S or W, and / or, the 287th amino acid residue is replaced with D, E, F, G, K, M or T, and / or, the 288th amino acid residue is replaced with A, F or R, and / or, the 289th amino acid residue is replaced with G or P, and / or, the 290th amino acid residue is replaced with I or Q, and / or, the 291st amino acid residue is replaced with A, and / or, the 292nd amino acid residue is replaced with E or G, and / or, the 293rd amino acid residue is replaced with F, K, N or P, and / or, the 294th amino acid residue is replaced with F, and / or, the 295th amino acid residue is replaced with L or N, and / or, the 296th amino acid residue is replaced with Q, and / or, the 297th amino acid residue is replaced with A, F, G, H, N or T, and / or, the 298th amino acid residue is replaced with E, and / or, the 299th amino acid residue is replaced with I, N or T, and / or, the 300th amino acid residue is replaced with P, Q or V, and / or, the 301st amino acid residue is replaced with C, and / or, the 302nd amino acid residue is replaced with H or N, and / or, the 303rd amino acid residue is replaced with F, T or V, and / or, the 304th amino acid residue is replaced with F, P or R, and / or, the 305th amino acid residue is replaced with H or K, and / or, the 309th amino acid residue is replaced with P, Q or S, and / or, the 310th amino acid residue is replaced with K or P, and / or, the 311th amino acid residue is replaced with A, E or Y, and / or, the 312th amino acid residue is replaced with E, H, K, L, M, V or Y, and / or, the 313th amino acid residue is replaced with E, P or T, and / or, the 314th amino acid residue is replaced with H, L or W, and / or, the 315th amino acid residue is replaced with G, P, V or Y, and / or, the 316th amino acid residue is replaced with E, F or S, and / or, the 317th amino acid residue is replaced with P, and / or, the 318th amino acid residue is replaced with F or R, and / or, the 320th amino acid residue is replaced with C, I or V, and / or, the 321st amino acid residue is replaced with E or Y, and / or,The 323rd amino acid residue is replaced with I, and / or, the 324th amino acid residue is replaced with E, M or T, and / or, the 325th amino acid residue is replaced with N, and / or, the 326th amino acid residue is replaced with L, and / or, the 327th amino acid residue is replaced with A, and / or, the 328th amino acid residue is replaced with P, S or Y, and / or, the 329th amino acid residue is replaced with A, F, M, N, Q or S, and / or, the 330th amino acid residue is replaced with R or W, and / or, the 331st amino acid residue is replaced with H, and / or, the 332nd amino acid residue is replaced with F, and / or, the 333rd amino acid residue is replaced with A or F, and / or, the 334th amino acid residue is replaced with D, E, F, P or W, and / or, the 335th amino acid residue is replaced with A, and / or, the 336th amino acid residue is replaced with P or Q, and / or, the 337th amino acid residue is replaced with I, P or V, and / or, the 338th amino acid residue is replaced with A, D or G, and / or, the 339th amino acid residue is replaced with L or T, and / or, the 340th amino acid residue is replaced with G or P, and / or, the 341st amino acid residue is replaced with E or T, and / or, the 342nd amino acid residue is replaced with C, P, Q, S or Y, and / or, the 343rd amino acid residue is replaced with C, K or M, and / or, the 344th amino acid residue is replaced with R or W, and / or, the 346th amino acid residue is replaced with N, and / or, the 349th amino acid residue is replaced with W, and / or, the 350th amino acid residue is replaced with K or S, and / or, the 351st amino acid residue is replaced with A, E, L, N or Q, and / or, the 352nd amino acid residue is replaced with M, and / or, the 353rd amino acid residue is replaced with N, Q or T, and / or, the 354th amino acid residue is replaced with Q, T or W, and / or, the 356th amino acid residue is replaced with H or I, and / or, the 357th amino acid residue is replaced with M, Q or R, and / or, the 361st amino acid residue is replaced with Y, and / or, the 362nd amino acid residue is replaced with K or W, and / or, the 363rd amino acid residue is replaced with D, G, I, P or Y, and / or, the 367th amino acid residue is replaced with H, N, R or V, and / or, the 368th amino acid residue is replaced with A, T, V, W or Y, and / or, the 369th amino acid residue is replaced with E, P, S or T, and / or, the 370th amino acid residue is replaced with R or V, and / or, the 371st amino acid residue is replaced with K or W, and / or, the 372nd amino acid residue is replaced with D or F, and / or, the 373rd amino acid residue is replaced with A, F, P or Y, and / or, the 375th amino acid residue is replaced with E or Q, and / or, the 376th amino acid residue is replaced with K, R or T, and / or, the 377th amino acid residue is replaced with R, and / or, the 379th amino acid residue is replaced with G, and / or,The 380th amino acid residue is replaced with E, P, or W, and / or the 382nd amino acid residue is replaced with C, and / or the 383rd amino acid residue is replaced with Q, R, T, or V, and / or the 384th amino acid residue is replaced with A, C, or Y, and / or the 386th amino acid residue is replaced with R, T, or W, and / or the 388th amino acid residue is replaced with N, P, or S, and / or the 389th amino acid residue is replaced with T, and / or the 390th amino acid residue is replaced with L or Y, and / or the 393rd amino acid residue is replaced with L, P, T, or W, and / or the 395th amino acid residue is replaced with E or K, and / or the 396th amino acid residue is replaced with E, K, L, N, R, S, or T; Preferably, the mutant is such that the 21st amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is replaced with K, and / or the 35th amino acid residue is replaced with M, and / or the 41st amino acid residue is replaced with Q, and / or the 42nd amino acid residue is replaced with I, and / or the 44th amino acid residue is replaced with Y, and / or the 55th amino acid residue is replaced with T, and / or the 56th amino acid residue is replaced with R or T, and / or the 64th amino acid residue is replaced with I, and / or the 73rd amino acid residue is replaced with A, and / or the 75th amino acid residue is replaced with L, and / or the 83rd amino acid residue is replaced with A, and / or the 87th amino acid residue is replaced with W, and / or the 92nd amino acid residue is replaced with P, and / or the 97th amino acid residue is replaced with R, and / or the 103rd amino acid residue is replaced with A, and / or the 114th amino acid residue is replaced with A, and / or the 119th amino acid residue is replaced with W, and / or the 123rd amino acid residue is replaced with P, and / or the 148th amino acid residue is replaced with F, and / or the 150th amino acid residue is replaced with K, and / or the 155th amino acid residue is replaced with I, and / or the 158th amino acid residue is replaced with C, and / or the 170th amino acid residue is replaced with N, and / or the 179th amino acid residue is replaced with T, and / or the 190th amino acid residue is replaced with T, and / or the 199th amino acid residue is replaced with F, and / or the 201st amino acid residue is replaced with M, and / or the 213th amino acid residue is replaced with V, and / or the 219th amino acid residue is replaced with P, and / or the 221st amino acid residue is replaced with W, and / or the 222nd amino acid residue is replaced with K, and / or the 229th amino acid residue is replaced with R, and / or the 242nd amino acid residue is replaced with W, and / or the 249th amino acid residue is replaced with W, and / or the 253rd amino acid residue is replaced with A, and / or the 257th amino acid residue is replaced with R or V, and / or the 261st amino acid residue is replaced with D, and / or the 284th amino acid residue is replaced with R, and / or the 287th amino acid residue is replaced with M, and / or the 297th amino acid residue is replaced with T, and / or the 299th amino acid residue is replaced with N, and / or the 304th amino acid residue is replaced with F, and / or the 315th amino acid residue is replaced with P, and / or the 318th amino acid residue is replaced with R, and / or the 344th amino acid residue is replaced with R, and / or the 356th amino acid residue is replaced with I, and / or the 370th amino acid residue is replaced with R, and / or the 373rd amino acid residue is replaced with A, and / or the 376th amino acid residue is replaced with T, and / or the 377th amino acid residue is replaced with R, and / or the 379th amino acid residue is replaced with G, and / orThe 388th amino acid residue is replaced with N, and / or, the 393rd amino acid residue is replaced with L, and / or, the 395th amino acid residue is replaced with K., 4. The preparation method of the compound shown in Formula I as described in claim 1, characterized in that, The mutant is any one of the following cases: (1) The mutant has differences in amino acid residues at least at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 297 and 315; preferably, the 297th amino acid residue is replaced by T, and the 315th amino acid residue is replaced by P. (2) The mutant has differences in amino acid residues at least at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 297, 315, 373 and 376; preferably, the 297th amino acid residue is replaced by T, the 315th amino acid residue is replaced by P, the 373rd amino acid residue is replaced by A, and the 376th amino acid residue is replaced by T. (3) The mutant has differences in amino acid residues at least at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 44, 75, 297 and 376; preferably, the 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 297th amino acid residue is replaced by T, and the 276th amino acid residue is replaced by T. (4) The mutant has differences in amino acid residues at least at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 35, 44, 75, 92, 150, 297, 315, 373, 376 and 393; preferably, the 35th amino acid residue is replaced by M, the 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 92nd amino acid residue is replaced by P, the 150th amino acid residue is replaced by K, the 297th amino acid residue is replaced by T, the 315th amino acid residue is replaced by P, the 373rd amino acid residue is replaced by A, the 376th amino acid residue is replaced by T, and the 393rd amino acid residue is replaced by L. (5) The mutant has differences in amino acid residues at least at the following sites on the amino acid sequence shown in SEQ ID NO: 1: positions 44, 56, 75, 92, 150, 257, 297, 315, 373, and 376; preferably, the amino acid residue at position 44 is replaced by Y, the amino acid residue at position 56 is replaced by R or T, the amino acid residue at position 75 is replaced by L, the amino acid residue at position 92 is replaced by P, the amino acid residue at position 150 is replaced by K, the amino acid residue at position 257 is replaced by V or R, the amino acid residue at position 297 is replaced by T, the amino acid residue at position 315 is replaced by P, the amino acid residue at position 373 is replaced by A, and the amino acid residue at position 376 is replaced by T.

5. The preparation method of the compound shown in Formula I as described in Claim 1, characterized in that, The differences of the mutant on the amino acid sequence shown in SEQ ID NO: 1 are shown in the following table:

6. The preparation method of the compound shown in Formula I as described in claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The compound represented by Formula II is The corresponding compound represented by Formula I is (2) The mass ratio of the enol reductase or its mutant to the compound shown in Formula II is (0.1 - 20):1, such as (0.25 - 4):1, and further such as 0.25:1, 0.4:1, 0.5:1, 1:1, or 2:1; (3) The solvent is a buffer solution, such as one or more selected from phosphate buffer solution, tris(hydroxymethyl)aminomethane buffer solution, and triethanolamine buffer solution. The pH of the buffer solution can be 5.5 - 9.5, such as 6.5, 7.0, 7.5, or 8.5; Preferably, the solvent is phosphate buffer solution with pH 6.5, phosphate buffer solution with pH 7.0, phosphate buffer solution with pH 7.5, phosphate buffer solution with pH 8.5, triethanolamine buffer solution (TEOA) with pH 6.5, or tris(hydroxymethyl)aminomethane buffer solution with pH 6.5; (4) The volume - mass ratio of the solvent to the compound shown in Formula II is 10 - 2000 mL / g, such as 10 - 200 mL / g, and further such as 14 mL / g, 35 mL / g, or 200 mL / g; (5) The reduction reaction further includes a co - solvent, such as DMSO; Preferably, the volume - mass ratio of the co - solvent to the compound shown in Formula II is 10 - 500 mL / g, such as 10 - 30 mL / g, and further such as 10 mL / g, 15 mL / g, or 30 mL / g; (6) The coenzyme is a reduced coenzyme, such as nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide, and preferably nicotinamide adenine dinucleotide phosphate; (7) In the reduction reaction, when the coenzyme is an oxidized coenzyme or its salt, or when the amount of the reduced coenzyme is not sufficient, the reduction reaction further includes a coenzyme regeneration system; the coenzyme regeneration system includes a reductase or dehydrogenase, and a hydrogen donor; Preferably, the coenzyme regeneration system is any one of the following systems: System 1: Carbonyl reductase (KRED), preferably ketoreductase or alcohol dehydrogenase (ADH), and a hydrogen donor such as isopropanol; System II: Glucose dehydrogenase (GDH), and a hydrogen donor such as glucose; (8) The reaction temperature of the reduction reaction is 0 to 37 °C, such as 4 °C, 7 °C, 17 °C, 18 °C, 20 °C or 27 °C; (9) The reaction time of the reduction reaction is 0.5 to 42 h, such as 4 to 24 h, such as 8 h, 16 h or 24 h.

7. The method for preparing the compound represented by formula I as described in claim 6, wherein the coenzyme is an oxidized coenzyme or a salt thereof. At this time, the reduction reaction includes a coenzyme regeneration system, and the coenzyme regeneration system includes a reductase or dehydrogenase, and a hydrogen donor.

8. The method for preparing the compound shown in Formula I as claimed in claim 6 or 7, characterized in that, In the reduction reaction, when the reduction reaction further includes a coenzyme regeneration system, at this time, the reduction reaction satisfies one or more of the following conditions: (1) The oxidized coenzyme or a salt thereof is nicotinamide adenine dinucleotide (NAD+), oxidized nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide disodium salt (NAD-Na2) or nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2), preferably oxidized nicotinamide adenine dinucleotide phosphate (NADP+) or nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2), such as nicotinamide adenine dinucleotide phosphate disodium salt (NADP-Na2); (2) The mass ratio of the reductase or dehydrogenase to the compound represented by formula II is (0.05 to 6):1, such as 0.05:1, 0.1:1 or 0.2:1; (3) The mass ratio of the reductase or dehydrogenase to the hydrogen donor is (0.05 to 0.2):1, such as 0.05:1, 0.1:1 or 0.2:1; (3) The mass ratio of the oxidized coenzyme to the compound represented by formula II is (0.001 to 0.5):1, such as (0.01 to 0.1):1, and further such as 0.05:1; (4) The coenzyme regeneration system includes a dehydrogenase and a hydrogen donor. The dehydrogenase is glucose dehydrogenase, and the hydrogen donor is glucose, such as D-glucose.

9. A mutant of an alkene reductase, which is any one of the following cases: (1) The mutant has at least the following differences in amino acid residues at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 297 and 315; preferably, the amino acid residue at position 297 is replaced by T, and the amino acid residue at position 315 is replaced by P; (2) The mutant has at least the following differences in amino acid residues at the following positions on the amino acid sequence shown in SEQ ID NO:1: positions 297, 315, 373 and 376; preferably, the amino acid residue at position 297 is replaced by T, the amino acid residue at position 315 is replaced by P, the amino acid residue at position 373 is replaced by A, and the amino acid residue at position 376 is replaced by T; (3) The mutant has at least the following differences in amino acid residues at the following sites on the amino acid sequence shown in SEQ ID NO: 1: the 44th, 75th, 297th, and 376th positions; wherein, The 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 297th amino acid residue is replaced by T, and the 276th amino acid residue is replaced by T; (4) The mutant described above has differences in amino acid residues at least at the following positions in the amino acid sequence shown in SEQ ID NO: 1: the 35th, 44th, 75th, 92nd, 150th, 297th, 315th, 373rd, 376th, and 393rd positions; preferably, the 35th amino acid residue is replaced by M, the 44th amino acid residue is replaced by Y, the 75th amino acid residue is replaced by L, the 92nd amino acid residue is replaced by P, the 150th amino acid residue is replaced by K, the 297th amino acid residue is replaced by T, the 315th amino acid residue is replaced by P, the 373rd amino acid residue is replaced by A, the 376th amino acid residue is replaced by T, and the 393rd amino acid residue is replaced by L; (5) The mutant described above has differences in amino acid residues at least at the following positions in the amino acid sequence shown in SEQ ID NO: 1: the 44th, 56th, 75th, 92nd, 150th, 257th, 297th, 315th, 373rd, and 376th positions; preferably, the 44th amino acid residue is replaced by Y, the 56th amino acid residue is replaced by R or T, the 75th amino acid residue is replaced by L, the 92nd amino acid residue is replaced by P, the 150th amino acid residue is replaced by K, the 257th amino acid residue is replaced by V or R, the 297th amino acid residue is replaced by T, the 315th amino acid residue is replaced by P, the 373rd amino acid residue is replaced by A, and the 376th amino acid residue is replaced by T; (6) The mutant 002 - mutant 213 as described in claim 5.

10. An isolated nucleic acid, wherein the nucleic acid encodes a mutant of the enoyl - reductase as described in claim 9.

11. A recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid as described in claim 10.

12. A transformant, wherein the transformant contains the recombinant expression vector as described in claim 11.

13. A method for preparing a mutant of enoyl - reductase, which comprises the following step: culturing the transformant as described in claim 12 and obtaining a culture containing the mutant of enoyl - reductase.

14. Use of the enoyl - reductase or its mutant as described in any one of claims 1 - 6 in the asymmetric reduction of prochiral olefins, wherein the prochiral olefin is the compound shown in formula II as described in any one of claims 1 - 6; Preferably, in the said use, the reaction operation and conditions of the asymmetric reduction are as described in the reduction reaction of any one of claims 1 - 6.

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