Bayer Williger monooxygenase mutant and application thereof

Abstract

The invention discloses a Bayer Williger monooxygenase mutant and an application of the Bayer Williger monooxygenase mutant. The invention provides a monooxygenase mutant, and compared with an amino acid sequence shown as SEQ ID NO: 1, an amino acid sequence of the monooxygenase mutant contains amino acid residue difference of one or more sites; the activity of the monooxygenase mutant is improved compared with that of Bayer-Williger monooxygenase with the amino acid sequence shown as SEQ ID NO: 1, wherein the amino acid sequence of the Bayer-Williger monooxygenase is shown as SEQ ID NO: 1. According to the monooxygenase mutant provided by the invention, the chiral thiosulfinate compound can be generated with relatively high yield and stereoselectivity, and the reaction condition is mild.

Description

Technical Field

[0001] This invention relates to a Bayer Welig monooxygenase mutant and its application, belonging to the fields of biotechnology and pharmaceutical intermediate synthesis. Background Technology

[0002] tert-butylsulfinamides, including both R- and S-type structures, are increasingly widely used in academia and industry as key chiral sources for the synthesis of chiral amine drugs and their intermediates. More and more drugs utilize this compound as a chiral amine adjuvant. For example, it can be used in the synthesis of drugs such as the allergy medication cetirizine hydrochloride, the anti-Parkinson's disease drug rasagiline, and the Alzheimer's disease treatment rivastigmine (Acc. Chem. Res. 2002, 35, 984 or CN106478471A / B). Tert-butylsulfinamides are mainly obtained through the chiral tert-butylsulfinyl tert-butyl thiol ester, which in turn is mainly obtained through the asymmetric oxidation of tert-butyl disulfide.

[0003] The existing synthetic methods for chiral tert-butyl sulfinyl tert-butyl thiol ester mainly involve oxidation with a chiral ligand via hydrogen peroxide under vanadium catalysis to generate chiral tert-butyl sulfinyl tert-butyl thiol ester (J.Am.Chem.Soc.1997,119,9913-9914 / ORGANIC LETTERS 2003 Vol.5,No.8 1317-1320 / CN106478471A / B).

[0004] Existing synthetic methods involve the oxidation of chiral tert-butyl sulfinyl tert-butyl thiol esters by hydrogen peroxide under vanadium catalysis. This process requires the addition of a metal catalyst and a chiral ligand, and involves the slow addition of hydrogen peroxide, making it complex and yielding a chiral product with insufficient ee (efficacy). Furthermore, no enzyme-catalyzed method for obtaining chiral tert-butyl sulfinyl tert-butyl thiol esters has been reported to date. Summary of the Invention

[0005] To address the lack of a technical method for preparing chiral tert-butylsulfinyl tert-butyl mercaptan ester using bio-enzymatic catalysis, this invention provides a Bayer Willigan monooxygenase mutant and its applications. The oxygenation reaction catalyzed by the Bayer Willigan monooxygenase of this invention operates under mild conditions, produces products with high chiral purity, and exhibits good mutant stereoselectivity and catalytic activity.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a monooxygenase mutant, wherein the amino acid sequence of the monooxygenase mutant differs from that of the amino acid sequence shown in SEQ ID NO:1 by one or more sites of amino acid residue difference; the activity of the monooxygenase mutant is increased relative to that of the Bayer Welig monooxygenase shown in SEQ ID NO:1.

[0008] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant differs from the amino acid sequence shown in SEQ ID NO:1 by one or more amino acid residues at the following sites: position 4, position 6, position 16, position 28, position 30, position 32, position 45, position 46, position 49, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 67, position 70, position 80, position 81, position 82, position 83, position 84, position 87, position 96, position 102, position 111, position 127, position 128, position 131, position 141, position 142, position 14 3rd, 144th, 145th, 146th, 151st, 153rd, 156th, 159th, 165th, 166th, 167th, 170th, 171st, 175th, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 200th, 207th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 222nd, 223rd, 226th, 227th, 230th, 234th, 236th 239th, 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 252nd, 259th, 261st, 269th, 272nd, 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 291st, 292nd, 307th, 310th, 316th, 321st, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 334th, 341st, 344th, 349th 359th, 364th, 368th, 370th, 377th, 379th, 380th, 381st, 382nd, 383rd, 389th, 396th, 401st, 405th, 406th, 426th, 427th, 430th, 431st, 432nd, 433rd, 434th, 435th, 436th, 437th, 438th, 439th, 442nd, 448th, 455th, 457th, 459th, 465th, 466th, 470th, 473rd, 477th, 480th, 481st, 488thThe 490th, 491st, 492nd, 497th, 501st, 504th, 505th, 506th, 507th, 522nd, 532nd, or 537th position.

[0009] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0010] (1) The 4th position: C, D, E, F, G, H, L, M, P, Q, R, S, V, W or Y;

[0011] (2) The 6th digit: A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0012] (3) The 16th character: A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V, W or Y;

[0013] (4) 28th position: C, D, F, G, H, I, L, N, P, Q, R, T, W or Y;

[0014] (5) The 30th character: A, C, D, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y;

[0015] (6) 32nd position: A, F, G, H, L, M, N, Q, R, T or W;

[0016] (7) 45th position: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, W or Y;

[0017] (8) 46th position: A, C, D, E, F, G, I, K, L, M, Q, R, S, T, V or Y;

[0018] (9) 49th position: D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0019] (10) 51st position: A, C, E, F, G, H, K, L, N, P, Q, R, T, V or W;

[0020] (11) 52nd position: A, C, D, F, G, H, I, M, N, Q, R, S, T, V or Y;

[0021] (12) 53rd position: A, C, D, F, H, I, K, L, M, P, Q, R, T or V;

[0022] (13) 54th position: C, D, E, F, H, I, K, P, Q, R, S, V, W or Y;

[0023] (14) 55th position: A, C, D, E, F, G, G, L, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0024] (15) Position 56: A, C, D, E, F, G, H, I, L, M, P, Q, S, T, W, Y;

[0025] (16) 57th position: A, C, E, F, G, H, I, K, L, M, P, Q, R, S, T, W or Y;

[0026] (17) 58th position: C, D, E, F, G, I, K, L, M, N, P, Q, R, S or W;

[0027] (18) 59th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0028] (19) 60th position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, V, W or Y;

[0029] (20) 61st position: A, C, D, E, F, G, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0030] (21) 62nd position: A, C, D, E, F, G, H, I, M, N, P, Q, S, T, V, W or Y;

[0031] (22) 63rd position: A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T or W;

[0032] (23) 64th position: A, D, F, G, H, K, L, M, P, Q, S, V, W or Y;

[0033] (24) 67th position: C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V or Y;

[0034] (25) 70th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;

[0035] (26) 80th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, V, W or Y;

[0036] (27) 81st position: A, D, E, F, G, H, K, L, N, P, Q, R, S, T, V or W;

[0037] (28) 82nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or W;

[0038] (29) 83rd position: A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, V, W or Y;

[0039] (30) 84th position: A, C, D, E, F, H, L, M, N, P, Q, R, S, V, W or Y;

[0040] (31) 87th position: A, D, I, K, L, N, P, Q, R, S, T or W;

[0041] (32) 96th position: A, D, F, G, K, L, M, N, Q, R or Y;

[0042] (33) 102nd position: A, C, D, E, F, G, H, I, M, P, Q, V or Y;

[0043] (34) 111th position: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y;

[0044] (35) 127th position: A, C, D, F, G, H, K, L, Q, R, S, W or Y;

[0045] (36) 128th position: A, C, D, E, G, H, I, L, M, N, P, Q, R, S, T, V or W;

[0046] (37) 131st position: D, E, G, H, I, L, P, V, W or Y;

[0047] (38) 141st position: A, C, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0048] (39) 142nd position: A, C, E, F, H, I, L, N, P, Q, R, S, T, V, W or Y;

[0049] (40) 143rd position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0050] (41) 144th position: A, F, G, H, K, M, N, P, Q, R, S, T, V or Y;

[0051] (42) 145th position: C, D, E, G, I, K, M, N, Q, T, V, W or Y;

[0052] (43) 146th position: C, D, F, G, H, K, L, M, N, P, Q, R, T, V or Y;

[0053] (44) 151st position: A, C, D, E, F, G, H, I, K, M, P, Q, R, S, T or V;

[0054] (45) 153rd position: C, D, E, F, G, I, L, M, N, P, Q, S, T, W or Y;

[0055] (46) 156th position: A, C, D, E, F, G, I, L, M, P, Q, R, S, T, V, W or Y;

[0056] (47) 159th position: A, D, E, F, G, H, I, L, M, N, P, Q, R, V, W or Y;

[0057] (48) 165th position: A, C, D, E, F, H, I, K, L, M, P, Q, R, S, V, W or Y;

[0058] (49) 166th position: C, D, E, F, G, H, I, K, L, M, Q, R, T or Y;

[0059] (50) 167th position: A, C, D, E, F, H, I, L, M, N, P, Q, S, T or Y;

[0060] (51) 170th position: C, D, E, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0061] (52) 171st position: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0062] (53) 175th position: C, D, F, H, M, N, P, Q, S, T, W or Y;

[0063] (54) 184th position: A, D, E, F, H, I, K, L, M, Q, S, V, W or Y;

[0064] (55) 185th position: A, C, D, E, F, I, K, L, N, Q, R, T, V, W or Y;

[0065] (56) 186th position: A, D, E, F, G, H, I, K, L, P, Q, R, T, V, W or Y;

[0066] (57) 187th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or W;

[0067] (58) 188th position: C, D, E, F, H, I, K, L, M, N, Q, R, S, T or Y;

[0068] (59) 189th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or W;

[0069] (60) 190th position: A, C, D, F, G, H, I, L, M, N, R, S, T, V, W or Y;

[0070] (61) 191st position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;

[0071] (62) 192nd position: A, C, D, E, F, H, I, K, L, N, P, Q, R, S, W or Y;

[0072] (63) 193rd position: A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, V or W;

[0073] (64) 200th position: C, D, E, F, H, I, M, N, P, Q, R, S, V or Y;

[0074] (65) 207th position: A, C, D, G, H, I, K, L, M, N, P, T, V, W or Y;

[0075] (66) 210th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0076] (67) 211th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W;

[0077] (68) 212th position: A, C, E, F, G, H, I, K, L, M, N, P, R, T, W or Y;

[0078] (69) 213th position: A, C, D, F, H, I, K, L, N, P, Q, R, T or Y;

[0079] (70) 214th position: A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T or V;

[0080] (71) 215th position: A, D, F, G, H, K, L, M, P, Q, R, S, T, V or Y;

[0081] (72) 216th position: A, C, D, H, I, K, M, N, P, Q, R, T, V, W or Y;

[0082] (73) 217th position: A, D, E, F, H, I, K, L, M, Q, R, S, T, V, W or Y;

[0083] (74) 222nd position: A, C, F, G, H, I, K, L, N, P, Q, R, S, T, W or Y;

[0084] (75) 223rd position: A, C, D, F, G, H, I, K, L, M, N, P, Q, T, V, W or Y;

[0085] (76) 226th position: A, D, E, F, G, I, L, M, N, Q, R, S, V, W or Y;

[0086] (77) 227th position: A, C, E, G, H, I, L, N, P, Q, R, T or V;

[0087] (78) 230th position: A, C, E, G, K, M, N, P, R, S, T, V, W or Y;

[0088] (79) 234th position: A, C, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0089] (80) 236th position: A, C, D, E, F, G, H, K, M, P, R, S, T, V or Y;

[0090] (81) 239th position: C, D, E, F, G, H, L, M, N, P, Q, R, S, T, W or Y;

[0091] (82) 242nd position: A, C, D, E, F, G, I, L, M, N, P, Q, R, T, V or Y;

[0092] (83) 243rd position: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or Y;

[0093] (84) 244th position: A, C, D, E, F, G, H, I, K, M, Q, R, S, T, V, W or Y;

[0094] (85) 245th position: C, D, E, F, G, H, L, M, N, P, Q, R, S, V, W or Y;

[0095] (86) 246th position: A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0096] (87) 247th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W;

[0097] (88) 248th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, W or Y;

[0098] (89) 249th position: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V or W;

[0099] (90) 250th position: A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0100] (91) 252nd position: A, C, D, G, H, I, K, L, M, Q, R, S, V or Y;

[0101] (92) 259th position: A, D, F, G, H, I, L, M, N, P, Q, R, T, W or Y;

[0102] (93) 261st position: C, D, G, H, I, K, L, P, R, S, T, V, W or Y;

[0103] (94) 269th position: A, C, D, E, H, I, L, M, P, Q, R, T, V, W or Y;

[0104] (95) 272nd position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, T, V or Y;

[0105] (96) 274th position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, T, V or W;

[0106] (97) 275th position: A, C, D, E, F, I, K, L, M, N, S, T, V, W or Y;

[0107] (98) 276th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0108] (99) 277th position: A, C, E, G, H, I, K, L, N, Q, R, S, T, V, W or Y;

[0109] (100) 278th position: A, C, D, E, F, H, I, L, P, Q, S, T, V, W or Y;

[0110] (101) 279th position: A, C, D, E, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0111] (102) 280th position: A, D, F, G, H, K, N, P, R, T, W or Y;

[0112] (103) 281st position: A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y;

[0113] (104) 291st position: A, C, E, H, I, K, L, P, Q, R, W or Y;

[0114] (105) 292nd position: A, C, F, G, H, I, L, M, Q, V or W;

[0115] (106) 307th position: A, C, D, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0116] (107) 310th position: C, D, E, G, L, M, N, P, Q, R, S, T, V, W or Y;

[0117] (108) 316th position: A, D, I, L, M, N, V, W or Y;

[0118] (109) 321st position: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V or Y;

[0119] (110) 326th position: A, C, D, F, G, H, L, M, N, P, Q, R, S, V or W;

[0120] (111) 327th position: A, C, D, F, G, H, K, L, M, N, P, S, T or Y;

[0121] (112) 328th position: A, D, F, G, I, K, L, N, P, R, T, V, W or Y;

[0122] (113) 329th position: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, W or Y;

[0123] (114) 330th position: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0124] (115) 331st position: A, C, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0125] (116) 332nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;

[0126] (117) 334th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, V or W;

[0127] (118) 341st position: A, C, E, F, G, K, L, M, N, Q, R, T, V, W or Y;

[0128] (119) 344th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y;

[0129] (120) 349th position: C, E, G, H, I, M, Q, S, T, V, W or Y;

[0130] (121) 359th position: A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0131] (122) 364th position: A, C, D, F, H, I, K, L, M, N, P, Q, R, S, V, W or Y;

[0132] (123) 368th position: A, C, D, E, H, I, K, L, M, N, Q, R, S, V, W or Y;

[0133] (124) 370th position: A, C, D, G, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0134] (125) 377th position: D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W;

[0135] (126) 379th position: C, D, F, H, I, K, L, M, N, P, R, S, T, V or W;

[0136] (127) 380th position: A, C, D, G, I, K, L, M, P, Q, R, S, T, W or Y;

[0137] (128) 381st position: A, E, F, G, I, K, L, M, N, P, Q, R, S, V, W or Y;

[0138] (129) 382nd position: C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W or Y;

[0139] (130) 383rd position: C, D, E, F, G, K, L, M, N, P, Q, R, S, T, W or Y;

[0140] (131) 389th position: A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V or W;

[0141] (132) 396th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0142] (133) 401st position: A, D, G, I, L, M, P, Q, R, S, T, V, W or Y;

[0143] (134) 405th position: C, E, F, H, I, L, M, R, T, V, W or Y;

[0144] (135) 406th position: A, D, F, G, H, I, K, L, M, P, Q, R, S, T, W or Y;

[0145] (136) 426th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W or Y;

[0146] (137) 427th position: A, C, D, E, F, H, K, L, M, N, P, Q, R, S, T, W or Y;

[0147] (138) 430th position: A, D, E, F, I, K, L, N, P, Q, R, S, V, W or Y;

[0148] (139) 431st position: D, E, F, G, H, I, K, L, M, N, Q, R, T, V or Y;

[0149] (140) 432nd position: A, C, D, G, H, I, K, L, M, N, P, R or Y;

[0150] (141) 433rd position: C, D, E, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y;

[0151] (142) 434th position: A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W or Y;

[0152] (143) 435th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0153] (144) 436th position: C, D, E, G, H, I, K, L, M, N, Q, R, S, V, W or Y;

[0154] (145) 437th position: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V or W;

[0155] (146) 438th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, V, W or Y;

[0156] (147) 439th position: A, C, D, E, F, G, H, K, L, M, N, P, Q, S, V, W or Y;

[0157] (148) 442nd position: A, C, E, F, G, H, I, K, L, M, N, P, R, S, T, W or Y;

[0158] (149) 448th position: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0159] (150) 455th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W;

[0160] (151) 457th position: A, C, D, E, F, H, I, L, P, Q, R, S, V, W or Y;

[0161] (152) 459th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0162] (153) 465th position: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0163] (154) 466th position: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0164] (155) 470th position: A, D, E, F, H, I, K, L, M, N, S, T, V or W;

[0165] (156) 473rd position: F, I, K, L, M, P, R or S;

[0166] (157) 477th position: A, C, D, E, G, L, M, R, S or Y;

[0167] (158) 480th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0168] (159) 481st position: A, C, D, E, F, G, H, I, N, P, Q, R, S, T, V, W or Y;

[0169] (160) 488th position: A, C, D, E, F, H, I, K, L, M, P, S, T, W or Y;

[0170] (161) 490th position: C, D, E, G, H, I, K, L, M, N, Q, R, T, V, W or Y;

[0171] (162) 491st position: A, C, D, E, F, H, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0172] (163) 492nd position: A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0173] (164) 497th position: A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y;

[0174] (165) 501st position: C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0175] (166) 504th position: A, D, E, H, I, K, M, N, P, Q, S, T or V;

[0176] (167) 505th position: A, C, D, E, G, I, K, L, N, P, R, T, V, W or Y;

[0177] (168) 506th position: A, C, D, F, G, H, K, L, M, N, Q, R, S, T or W;

[0178] (169) 507th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0179] (170) 522nd position: A, D, E, F, G, H, I, K, L, M, P, Q, S, T, W or Y;

[0180] (171) 532nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or Y;

[0181] (172) 537th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y.

[0182] In some embodiments of the present invention, the activity is the catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II or compound III as shown in structure III.

[0183]

[0184] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared to the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] In the above mutants, the " / " between each site indicates that the amino acid sequence of the mutant includes the differences in amino acid residues at these sites compared with the amino acid sequence shown in SEQ ID NO:1.

[0193] In some embodiments of the present invention, the monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II.

[0194] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared to the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue differences: 055I / S / T / V, 111C / N / Y, 143D / F / P / R, 210E / H / Q / R / S, 246G / L / M / P, 276D / S / W, 435A / C / I / S / T, 438A / H / M / Y, 448G / L / R, 459D / I / K / Q, and 537F / I / N / S. In this invention, the " / " in the amino acid residue differences at the same site (e.g., 055I / S / T / V) indicates an "OR" relationship between the mutagenic amino acid residues at that site, and similar descriptions are used for other sites.

[0195] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further comprises one or more differences selected from the following amino acid residues: 004H, 006N / T, 045C / V, 059I / L / M / Y, 082A / C / P / S, 083S, 096L / Y, 131I, 165K / Q, 188I, 193V, 214G, 223M / P, 250G, 307H, 330E / Q, 368S, 370G, 380G / K, 381W, 396N / V / Y, 433H / S / V, 455E / N / P / T, 480C / D / V, and 501I / K / V / W.

[0196] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further includes the following combinations of amino acid residue differences: 433H / S / V, 455E / N / P / T, 480C / D / V, and 501I / K / V / W, and one or more selected from the following amino acid residue differences: 004H, 006N / T, 045C / V, 059I / L / M / Y, 082A / C / P / S, 083S, 096L / Y, 131I, 165K / Q, 188I, 193V, 214G, 223P, 250G, 307H, 330E / Q, 368S, 370G, 380G / K, 381W, and 396V / Y.

[0197] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared to the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0198]

[0199] In other embodiments of the invention, the monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to compound II as shown in structure II, and the yield of compound II as shown in structure II is greater than 90%.

[0200] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared to the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] In the above mutants, the " / " between each site indicates that the amino acid sequence of the mutant includes the differences in amino acid residues at these sites compared with the amino acid sequence shown in SEQ ID NO:1.

[0210] In some embodiments of the present invention, the monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to obtain compound III as shown in structure III.

[0211] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, includes the following amino acid residue differences: 191C / D / F / G / H / I / K / P / S / W / Y, 329C / I / K / M / R / S / T / V / W / Y, 332C / E / F / H / I / L / M / N / Q / T, 344D / E / F / H / K / M / N / R / T / W, 426D / E / I / N / P / Q / R / S / T / V / W / Y, and 532A / D / E / G / K / P / Q / W / Y.

[0212] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further comprises one or more differences selected from the following amino acid residues: 004H, 006T, 016I / L / N / W / Y, 030D / G / H / Q / R, 055I / K / T / W, 067E / F / I / M / N / P / S / V, 070D / G / K / N / Q / S / T, 083C / D / E / F / I / K / R / S / T, 096L / Y, 111A / D / Y, 141H / P / Q / V / W / Y, 143F / P / R / V, 165K / Q, 185A / C / E / F / H / I, 223M / P, 227E / L / Q / V, 246G, 250G, 259A / C / E / G / R / V, 276D / N, 307H, 368S, 370G, 380G / K, 381A / C / G / K / M / T / V / Y, 435C / F, 438H / Y, 442H / I / S / Y, 448A / R, 459D / I, 497A / C / M / Q / R / V, 501I / K / Q / R / V, 504A / F / H / N / S / T / V, 506E / I / Q / R / S, 522A / F / G / I / R / S / Y and 537F / L / N.

[0213] In some embodiments of the present invention, the amino acid sequence of the monooxygenase mutant, compared to the amino acid sequence shown in SEQ ID NO:1, includes one or more differences selected from the following amino acid residues:

[0214]

[0215]

[0216] In other embodiments of the invention, the monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to compound III as shown in structure III, and the yield of compound III as shown in structure III is greater than 90%.

[0217] A second aspect of the present invention provides an isolated nucleic acid that encodes a monooxygenase mutant as described in the first aspect of the present invention.

[0218] A third aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the second aspect of the present invention.

[0219] A fourth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention.

[0220] In some embodiments of the present invention, the host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells, preferably Escherichia coli cells.

[0221] A fifth aspect of the present invention provides a method for preparing a monooxygenase mutant, the method comprising: culturing a transformant as described in the fourth aspect of the present invention to obtain the monooxygenase mutant.

[0222] A sixth aspect of the present invention provides an enzyme preparation containing a monooxygenase mutant as described in the first aspect of the present invention.

[0223] In some embodiments of the present invention, the enzyme preparation is a liquid preparation or a lyophilized powder.

[0224] A seventh aspect of the present invention provides an enzyme composition comprising Bayer Willig monooxygenase or a monooxygenase mutant as described in the first aspect of the present invention, and one or more of a coenzyme, an oxidoreductase, and a catalase.

[0225] In some embodiments of the present invention, the Bayer Willig monooxygenase is derived from Acinetobacter johnsonii, and its amino acid sequence is preferably shown as SEQ ID NO:1.

[0226] The eighth aspect of the present invention provides the use of Bayer Willig monooxygenase, monooxygenase mutants as described in the first aspect of the present invention, isolated nucleic acids as described in the second aspect of the present invention, recombinant expression vectors as described in the third aspect of the present invention, transformants as described in the fourth aspect of the present invention, enzyme preparations as described in the sixth aspect of the present invention, and / or enzyme compositions as described in the seventh aspect of the present invention in the catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II or compound III as shown in structure III.

[0227]

[0228] In some embodiments of the present invention, the Bayer Willig monooxygenase is derived from Acinetobacter johnsonii, and its amino acid sequence is preferably shown as SEQ ID NO:1.

[0229] The ninth aspect of the present invention provides a method for preparing compound II as shown in structure II or compound III as shown in structure III, the method comprising: contacting and reacting Bayer Willig monooxygenase, a monooxygenase mutant as described in the first aspect of the present invention, an enzyme preparation as described in the sixth aspect of the present invention, and / or an enzyme composition as described in the seventh aspect of the present invention with compound I as shown in structure I to obtain compound II or compound III.

[0230]

[0231] In some embodiments of the present invention, the method includes the following steps: in the presence of an organic solvent, a buffer solution, an oxidant, and catalase, the Bayer Welig monooxygenase or the monooxygenase mutant is contacted with and reacted with compound I to obtain compound II or compound III. In this reaction, catalase can eliminate the hydrogen peroxide produced in the reaction, avoiding or mitigating the effect on the production of Bayer Welig monooxygenase.

[0232] In some embodiments of the invention, the reaction further includes the use of a coenzyme, which includes oxidized coenzyme and reduced coenzyme.

[0233] In some specific embodiments of the present invention, the reaction further includes the step of regenerating the oxidized coenzyme into the reduced coenzyme.

[0234] In some specific embodiments of the present invention, the reduced coenzyme is any one or more of NADH and NADPH, and the oxidized coenzyme is NADPH. + and NADP + Any one or more of the following.

[0235] In some specific embodiments of the present invention, the regeneration of the oxidized coenzyme into the reduced coenzyme uses NAD. + NADP + NAD + and NADP + Any one of them.

[0236] In some embodiments of the present invention, the reaction further includes a hydrogen donor and an oxidoreductase.

[0237] In some embodiments of the invention, the hydrogen donor is isopropanol, and / or the oxidoreductase is KRED, for example, KRED derived from Lactobacillus kefir DSM 20587 (Genebank ID: WP_054768785).

[0238] In some embodiments of the present invention, the oxidant is air, a mixture of oxygen and nitrogen, a mixture of oxygen and argon, or a mixture of oxygen and helium; in the present invention, the reaction conditions are open, and the oxidant is air (especially oxygen).

[0239] In some embodiments of the present invention, the organic solvent may be a conventional organic solvent for such reactions in the art, preferably selected from one or more of ether solvents, epoxy solvents, ester solvents, sulfoxide solvents, amide solvents, and alcohol solvents; the sulfoxide solvent is, for example, dimethyl sulfoxide; the alcohol solvent is, for example, selected from one or more of methanol, ethanol, isopropanol, and n-butanol;

[0240] Preferably, the organic solvent is dimethyl sulfoxide.

[0241] In some embodiments of the present invention, the buffer solution may be a buffer solution conventional for such reactions in the art; preferably, the buffer solution is selected from one or more of sodium tetraborate buffer, phosphate buffer, triethanolamine buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0242] In some embodiments of the present invention, the Bayer Willig monooxygenase is derived from Acinetobacter johnsonii, and its amino acid sequence is preferably shown in SEQ ID NO:1.

[0243] In some embodiments of the present invention, the conditions of the method are selected from one or more of the following:

[0244] (1) The concentration of the buffer solution is 40-60 mM; preferably, the concentration of the buffer solution is 50 mM.

[0245] (2) The pH value of the buffer solution is 6.0 to 10.5, preferably 7.0 to 10.0, for example 7.0, 7.5, 8.0 or 8.5;

[0246] (3) The volume-to-mass ratio of the buffer solution to compound I is (10-200) mL / g, preferably (30-100) mL / g;

[0247] (4) The mass ratio of the Bayer Willig monooxygenase or the monooxygenase mutant to the compound I is (0.01-2):1, preferably (0.05-0.5):1, for example 0.1:1 or 0.2:1;

[0248] (5) The volume-to-mass ratio of the organic solvent to compound I is (1-50) mL / g, preferably (5-20) mL / g, and more preferably 10 mL / g;

[0249] (6) The mass ratio of the catalase to the compound I is (0.001-0.1):1, preferably (0.01-0.05):1, and more preferably 0.04:1;

[0250] (7) The mass ratio of the coenzyme to compound I is (0.001-0.1):1, preferably (0.01-0.05):1, and more preferably 0.04:1;

[0251] (8) The volume-to-mass ratio of the hydrogen donor to compound I is (1-10) mL / g, preferably 2 mL / g;

[0252] (9) The mass ratio of the oxidoreductase to compound I is (0.1-1):1, preferably 0.12:1;

[0253] (10) The reaction temperature is 20 to 40°C, preferably 20°C, 30°C or 40°C;

[0254] (11) The reaction time is 16 to 48 hours, preferably 18 hours.

[0255] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0256] The reagents and raw materials used in this invention are all commercially available.

[0257] The positive and progressive effects of this invention are as follows:

[0258] (1) The present invention uses genetic engineering to perform directed evolution of a Bayer Willig monooxygenase to obtain several mutants with high activity and stereospecificity. Using compound I as a substrate, the Bayer Willig monooxygenase mutants can generate chiral thiosulfinate compounds with high yield and stereoselectivity.

[0259] (2) The oxygenation reaction catalyzed by Bayer Willig monooxygenase in this invention is carried out under mild conditions. Detailed Implementation

[0260] Unless otherwise specified, the abbreviations of amino acids used in this invention are conventional in the field, and the specific abbreviations correspond to the amino acids shown in Table 1.

[0261] Table 1

[0262]

[0263] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are used to further illustrate the invention but should not be construed as limiting it. The Bayer Willigan monooxygenase of the present invention can be expressed and purified using recombinant expression technology or prepared through complete artificial synthesis. These methods are conventional means of obtaining enzyme proteins well-known in the art. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or according to the product instructions.

[0264]

[0265] The buffer formulation used in this embodiment of the invention is as follows:

[0266] The formula for a 50 mmol / L pH 7.0 sodium tetraborate buffer (Na₂B₄O₇ buffer) (sodium borate buffer; containing 1 M / L isopropylamine) is as follows: Sodium tetraborate decahydrate (MW 381.37): 19.06 g, isopropylamine: 86 mL, adjust pH to 7.0 with hydrochloric acid (at room temperature), and add water to a final volume of 1 L. To prepare different pH values, simply adjust the solution accordingly.

[0267] The formula for a 50 mmol / L pH 7.0 potassium phosphate buffer (PBS buffer) (containing 1 M / L isopropylamine) is as follows: Dissolve 2.654 g of KH₂PO₄ (MW: 136.09), 6.96 g of K₂HPO₄·3H₂O (MW: 228.22), and 86 mL of isopropylamine. Adjust the pH to 7.0 with hydrochloric acid (at room temperature), and add water to a final volume of 1 L. To prepare different pH values, adjust accordingly.

[0268] The formula for a 50 mmol / L pH 7.0 triethanolamine buffer (TEOA buffer) (containing 1 M / L isopropylamine) is as follows: Dissolve 7.46 g of triethanolamine (MW: 149.19), 86 mL of isopropylamine, adjust the pH to 7.0 with hydrochloric acid (at room temperature), and add water to a final volume of 1 L. To prepare buffers with different pH values, adjust the pH accordingly.

[0269] The formula for a 50 mmol / L pH 7.0 tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris buffer) (containing 1 M / L isopropylamine) is as follows: Dissolve 7.88 g of tris(hydroxymethyl)aminomethane hydrochloride (MW: 157.6), 86 mL of isopropylamine, adjust the pH to 7.0 (room temperature) with hydrochloric acid, and add water to a final volume of 1 L. To prepare buffers with different pH values, adjust the solution accordingly.

[0270] Construction of the Bayer Willigan monooxygenase mutant library:

[0271] Bayer Wellig monooxygenase SEQ ID NO:1:

[0272] MSQKMDFDAIVIGGGGFGGLYAVKKLRDELELKVQAFDKATDVAGTWYWNRYPGALTDTETHLYCYSWDKELLQSLEIKKKYVQGPDVRKYLQQVAEKHDLKKSYQFNTAVQSAHYNEADALWEVTTEYGDKYTARFLITALGLLSAPNLPNIKGINQFKGELHHTSRWPDDVSFEGKRVGVIGTGSTGVQVITAVAPLAKHLTVFQRSAQYSVPIGNDPLSEEDVKKIKDNYDKIWDGVWNSALAFGLNESTVPAMSVSAEERKAVFEKAW QTGGGFRFMFETFGDIATNMEANIEAQNFIKKGKIAEIVKDPAIAQKLMPQDLYAKRPLCDSGYYNTFNRDNVRLEDVKANPIVEITENGVKLENGDFVELDMLICATGFDAVDGNYVRMDIQGKNGLAMKDYWKEG PSSYMGVTVNNYPNMFMVLGPNGPFTNLPPSIESQVEWISDTIQYTVENNVESIEEATKEAEEQWTQTCANIAEMTLFPKAQSWIFGANIPGKKNTVYFYLGGLKEYRSALANCKNHAYEGFDIQLQRSDIKQPANA

[0273] The mutation sites of the mutant are selected from positions 4, 6, 16, 28, 30, 32, 45, 46, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 67, 70, 80, 81, 82, 83, 84, 87, 96, 102, 111, 127, 128, 131, 141, 142, 143, 144, 145, 146, and 151 of the amino acid sequence shown in SEQ ID NO:1. 153rd, 156th, 159th, 165th, 166th, 167th, 170th, 171st, 175th, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 200th, 207th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 222nd, 223rd, 226th, 227th, 230th, 234th, 236th, 239th, 242nd, 243rd, 244th, 245th 246th, 247th, 248th, 249th, 250th, 252nd, 259th, 261st, 269th, 272nd, 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 291st, 292nd, 307th, 310th, 316th, 321st, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 334th, 341st, 344th, 349th, 359th, 364th, 368th, 370th, 377th, 3rd 79th, 380th, 381st, 382nd, 383rd, 389th, 396th, 401st, 405th, 406th, 426th, 427th, 430th, 431st, 432nd, 433rd, 434th, 435th, 436th, 437th, 438th, 439th, 442nd, 448th, 455th, 457th, 459th, 465th, 466th, 470th, 473rd, 477th, 480th, 481st, 488th, 490th, 491st, 492nd, 497th, 501st, 504thThe 505th, 506th, 507th, 522nd, 532nd, and 537th positions.

[0274] Mutants with high catalytic activity, namely mutant 1-mutant 400, were screened out. The specific mutation sites and types are shown in Tables 2 and 3.

[0275] Example 1

[0276] In TEOA buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. The specific experimental steps are as follows:

[0277] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 100 mL 0.05 mol / L TEOA buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask, then add 1 g of compound I and 0.1 g of mutant 191. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract it with 10 mL of methanol. After extraction, remove the organic phase and distill it under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.07 g of product, with a yield of 98.2% and an isomer excess (ee) of 99.1%.

[0278]

[0279] The mutants in Example 1 were replaced with mutants 1 through 200, with all other reaction conditions remaining the same. The reaction results are shown in Table 2.

[0280] Table 2 Mutation types of mutants and their corresponding yields and isoform excess values.

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] Example 2

[0292] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0293] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 60 mL TEOA buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 42 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.1 g of Bayer Willigan monooxygenase with mutant 191 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.057 g of product, with a yield of 97.0% and an isomer excess (ee) of 98.2%.

[0294] Example 3

[0295] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0296] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 200 mL 0.05 mol / L TEOA buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 112 mL of the solution from the beaker to a 250 mL reaction flask, and add 1 g of compound I and 0.1 g of Bayer Willigan monooxygenase with mutant 191 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, remove 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.083 g of product, with a yield of 99.4% and an isomer excess (ee) of 99.7%.

[0297] Example 4

[0298] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0299] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L TEOA buffer (pH 7.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.1 g of Bayer Welig monooxygenase with mutant 74 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.076 g of product, with a yield of 98.7% and an isomer excess (ee) of 99.4%.

[0300] Example 5

[0301] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0302] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL TEOA buffer (pH 7.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.1 g of Bayer Willigan monooxygenase with mutant 81 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.059 g of product, with a yield of 97.2% and an isomer excess (ee) of 98.2%.

[0303] Example 6

[0304] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0305] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 100 mL 0.05 mol / L TEOA buffer (pH 7.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.1 g of Bayer Welig monooxygenase with mutant 120 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.993 g of product, with a yield of 91.1% and an isomer excess (ee) of 96.7%.

[0306] Example 7

[0307] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0308] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL TEOA buffer (pH 8.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 30 to the solution. Vortex to mix the resulting solution and react at 20 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.013 g of product, with a yield of 93.0% and an isomer excess (ee) of 97.6%.

[0309] Example 8

[0310] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0311] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL pH 8.0 TEOA buffer to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask, then add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 84. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.982 g of product, with a yield of 90.1% and an isomer excess (ee) of 91.3%.

[0312] Example 9

[0313] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0314] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL TEOA buffer (pH 8.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.2 g of Bayer Willigan monooxygenase with mutant 125 to the solution. Vortex to mix the resulting solution and react at 40 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.966 g of product, with a yield of 88.6% and an isomer excess (ee) of 90.5%.

[0315] Example 10

[0316] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0317] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L TEOA buffer (pH 8.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask, then add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 50. Vortex to mix the resulting solution and react at 20 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.994 g of product, with a yield of 91.2% and an isomer excess (ee) of 95.6%.

[0318] Example 11

[0319] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0320] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L TEOA buffer (pH 8.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.2 g of Bayer Willigan monooxygenase with mutant 95 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.962 g of product, with a yield of 88.3% and an isomer excess (ee) of 92.4%.

[0321] Example 12

[0322] In TEOA buffer, Bayer Wellig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound II from compound I. Specific experimental procedures:

[0323] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L TEOA buffer (pH 8.5) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask, then add 1 g of compound I and 0.2 g of Bayer Willigan monooxygenase with mutant 193. Vortex to mix the resulting solution and react at 40 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 0.952 g of product, with a yield of 87.4% and an isomer excess (ee) of 90.1%.

[0324] Example 13

[0325] In TEOA buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental procedures:

[0326] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL TEOA buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 62 mL of the solution from the beaker to a 100 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 290 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, remove 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.006 g of product, with a yield of 92.3% and an isomer excess (ee) of 97.2%.

[0327]

[0328] The mutants in Example 13 were replaced with mutants 201-400, with all other reaction conditions remaining the same. The reaction results are shown in Table 3.

[0329] Table 3 Mutation types of mutants and their corresponding yields and isoform excess values.

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339] Example 14

[0340] In TEOA buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental procedures:

[0341] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 200 mL 0.05 mol / L TEOA buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 112 mL of the solution from the beaker to a 100 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 290 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, remove 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.025 g of product, with a yield of 94.1% and an isomer excess (ee) of 98.1%.

[0342] Example 15

[0343] In PBS buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0344] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 200 mL 0.05 mol / L PBS buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 112 mL of the solution from the beaker to a 250 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 321 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, remove 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.076 g of product, with a yield of 98.7% and an isomer excess (ee) of 99.7%.

[0345] Example 16

[0346] In PBS buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0347] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL pH 7.0 PBS buffer to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask, then add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 321. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.000 g of product, with a yield of 91.8% and an isomer excess (ee) of 99.1%.

[0348] Example 17

[0349] In Tris buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0350] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 200 mL of 0.05 mol / L Tris buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 112 mL of the solution from the beaker to a 250 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 223 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, remove 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.054 g of product, with a yield of 96.7% and an isomer excess (ee) of 99.2%.

[0351] Example 18

[0352] In Tris buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0353] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 0.05 mol / L, 100 mL Tris buffer (pH 7.0) to a 500 mL beaker, and stir until a homogeneous solution of NADP+ and IPA is obtained. Take 62 mL of the solution from the beaker and add it to a 100 mL reaction flask. Add 1 g of compound I and 0.2 g of Bayer Welig monooxygenase with mutant 223 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction, take 1.0 mL of the reaction solution and extract with 10 mL of methanol. After extraction, remove the organic phase and distill under reduced pressure at -0.09 MPa (gauge pressure) to obtain 1.033 g of product, with a yield of 94.8% and an isomer excess (ee) of 98.5%.

[0354] Example 19

[0355] In Na₂B₄O₇ buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0356] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 200 mL of 0.05 mol / L Na2B4O7 buffer (pH 7.0) to a 500 mL beaker. Stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 112 mL of the solution from the beaker to a 250 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Willigan monooxygenase with mutant 278 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction was completed, 1.0 mL of the reaction solution was taken out and extracted with 10 mL of methanol. After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum of -0.09 MPa (gauge pressure) to finally obtain 1.080 g of product, with a yield of 99.1% and an isomer excess value (ee) of 99.6%.

[0357] Example 20

[0358] In Na₂B₄O₇ buffer, Bayer Welig monooxygenase catalyzed the synthesis of chiral thiosulfinate compound III from compound I. Specific experimental steps:

[0359] Add 40 mg NADP+, 120 mg KRED, 40 mg Catalase, 4 mL IPA (isopropanol), 20 mL DMSO (dimethyl sulfoxide), and 100 mL of 0.05 mol / L Na2B4O7 buffer (pH 7.0) to a 500 mL beaker. Stir until a homogeneous solution of NADP+ and IPA is obtained. Transfer 62 mL of the solution from the beaker to a 100 mL reaction flask, and add 1 g of compound I and 0.2 g of Bayer Willigan monooxygenase with mutant 278 to the solution. Vortex to mix the resulting solution and react at 30 °C for 18 h. After the reaction was completed, 1.0 mL of the reaction solution was taken out and extracted with 10 mL of methanol. After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum of -0.09 MPa (gauge pressure) to finally obtain 1.070 g of product, with a yield of 98.2% and an isomer excess value (ee) of 99.4%.

[0360] The above embodiments include preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A monooxygenase mutant, characterized in that, The amino acid sequence of the monooxygenase mutant contains one or more amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:1; the activity of the monooxygenase mutant is increased relative to the Bayer Welig monooxygenase shown in SEQ ID NO:

1.

2. The monooxygenase mutant as described in claim 1, characterized in that, The amino acid sequence of the monooxygenase mutant differs from the amino acid sequence shown in SEQ ID NO:1 by one or more amino acid residues at the following sites: position 4, position 6, position 16, position 28, position 30, position 32, position 45, position 46, position 49, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 67, position 70, position 80, position 81, position 82, position 83, position 84, position 87, position 96, position 102, position 111, position 127, position 128, position 131, position 141, position 142, position 143. 144th, 145th, 146th, 151st, 153rd, 156th, 159th, 165th, 166th, 167th, 170th, 171st, 175th, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 200th, 207th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 222nd, 223rd, 226th, 227th, 230th, 234th, 236th, 239th 242nd, 243rd, 244th, 245th, 246th, 247th, 248th, 249th, 250th, 252nd, 259th, 261st, 269th, 272nd, 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 291st, 292nd, 307th, 310th, 316th, 321st, 326th, 327th, 328th, 329th, 330th, 331st, 332nd, 334th, 341st, 344th, 349th, 359th, 3rd 64th, 368th, 370th, 377th, 379th, 380th, 381st, 382nd, 383rd, 389th, 396th, 401st, 405th, 406th, 426th, 427th, 430th, 431st, 432nd, 433rd, 434th, 435th, 436th, 437th, 438th, 439th, 442nd, 448th, 455th, 457th, 459th, 465th, 466th, 470th, 473rd, 477th, 480th, 481st, 488th, 490th, 491stThe 492nd, 497th, 501st, 504th, 505th, 506th, 507th, 522nd, 532nd, or 537th position.

3. The monooxygenase mutant as described in claim 2, characterized in that, The amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more differences selected from the following amino acid residues: (1) The 4th position: C, D, E, F, G, H, L, M, P, Q, R, S, V, W or Y; (2) The 6th digit: A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (3) The 16th character: A, C, D, E, G, H, I, K, L, M, P, Q, S, T, V, W or Y; (4) 28th position: C, D, F, G, H, I, L, N, P, Q, R, T, W or Y; (5) The 30th character: A, C, D, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; (6) 32nd position: A, F, G, H, L, M, N, Q, R, T or W; (7) 45th position: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, W or Y; (8) 46th position: A, C, D, E, F, G, I, K, L, M, Q, R, S, T, V or Y; (9) 49th position: D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; (10) 51st position: A, C, E, F, G, H, K, L, N, P, Q, R, T, V or W; (11) 52nd position: A, C, D, F, G, H, I, M, N, Q, R, S, T, V or Y; (12) 53rd position: A, C, D, F, H, I, K, L, M, P, Q, R, T or V; (13) 54th position: C, D, E, F, H, I, K, P, Q, R, S, V, W or Y; (14) Position 55: A, C, D, E, F, G, GL, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (15) Position 56: A, C, D, E, F, G, H, I, L, M, P, Q, S, T, W, Y; (16) 57th position: A, C, E, F, G, H, I, K, L, M, P, Q, R, S, T, W or Y; (17) 58th position: C, D, E, F, G, I, K, L, M, N, P, Q, R, S or W; (18) 59th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (19) 60th position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, V, W or Y; (20) 61st position: A, C, D, E, F, G, K, L, M, N, P, Q, R, S, T, V, W or Y; (21) 62nd position: A, C, D, E, F, G, H, I, M, N, P, Q, S, T, V, W or Y; (22) 63rd position: A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T or W; (23) 64th position: A, D, F, G, H, K, L, M, P, Q, S, V, W or Y; (24) 67th position: C, D, F, G, H, I, K, L, M, N, Q, R, S, T, V or Y; (25) 70th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; (26) 80th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, V, W or Y; (27) 81st position: A, D, E, F, G, H, K, L, N, P, Q, R, S, T, V or W; (28) 82nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or W; (29) 83rd position: A, C, D, E, F, G, H, I, K, L, N, P, R, S, T, V, W or Y; (30) 84th position: A, C, D, E, F, H, L, M, N, P, Q, R, S, V, W or Y; (31) 87th position: A, D, I, K, L, N, P, Q, R, S, T or W; (32) 96th position: A, D, F, G, K, L, M, N, Q, R or Y; (33) 102nd position: A, C, D, E, F, G, H, I, M, P, Q, V or Y; (34) 111th position: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y; (35) 127th position: A, C, D, F, G, H, K, L, Q, R, S, W or Y; (36) 128th position: A, C, D, E, G, H, I, L, M, N, P, Q, R, S, T, V or W; (37) 131st position: D, E, G, H, I, L, P, V, W or Y; (38) 141st position: A, C, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (39) 142nd position: A, C, E, F, H, I, L, N, P, Q, R, S, T, V, W or Y; (40) 143rd position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (41) 144th position: A, F, G, H, K, M, N, P, Q, R, S, T, V or Y; (42) 145th position: C, D, E, G, I, K, M, N, Q, T, V, W or Y; (43) 146th position: C, D, F, G, H, K, L, M, N, P, Q, R, T, V or Y; (44) 151st position: A, C, D, E, F, G, H, I, K, M, P, Q, R, S, T or V; (45) 153rd position: C, D, E, F, G, I, L, M, N, P, Q, S, T, W or Y; (46) 156th position: A, C, D, E, F, G, I, L, M, P, Q, R, S, T, V, W or Y; (47) 159th position: A, D, E, F, G, H, I, L, M, N, P, Q, R, V, W or Y; (48) 165th position: A, C, D, E, F, H, I, K, L, M, P, Q, R, S, V, W or Y; (49) 166th position: C, D, E, F, G, H, I, K, L, M, Q, R, T or Y; (50) 167th position: A, C, D, E, F, H, I, L, M, N, P, Q, S, T or Y; (51) 170th position: C, D, E, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (52) 171st position: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (53) 175th position: C, D, F, H, M, N, P, Q, S, T, W or Y; (54) 184th position: A, D, E, F, H, I, K, L, M, Q, S, V, W or Y; (55) 185th position: A, C, D, E, F, I, K, L, N, Q, R, T, V, W or Y; (56) 186th position: A, D, E, F, G, H, I, K, L, P, Q, R, T, V, W or Y; (57) 187th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or W; (58) 188th position: C, D, E, F, H, I, K, L, M, N, Q, R, S, T or Y; (59) 189th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or W; (60) 190th position: A, C, D, F, G, H, I, L, M, N, R, S, T, V, W or Y; (61) 191st position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; (62) 192nd position: A, C, D, E, F, H, I, K, L, N, P, Q, R, S, W or Y; (63) 193rd position: A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, V or W; (64) 200th position: C, D, E, F, H, I, M, N, P, Q, R, S, V or Y; (65) 207th position: A, C, D, G, H, I, K, L, M, N, P, T, V, W or Y; (66) 210th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (67) 211th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W; (68) 212th position: A, C, E, F, G, H, I, K, L, M, N, P, R, T, W or Y; (69) 213th position: A, C, D, F, H, I, K, L, N, P, Q, R, T or Y; (70) 214th position: A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T or V; (71) 215th position: A, D, F, G, H, K, L, M, P, Q, R, S, T, V or Y; (72) 216th position: A, C, D, H, I, K, M, N, P, Q, R, T, V, W or Y; (73) 217th position: A, D, E, F, H, I, K, L, M, Q, R, S, T, V, W or Y; (74) 222nd position: A, C, F, G, H, I, K, L, N, P, Q, R, S, T, W or Y; (75) 223rd position: A, C, D, F, G, H, I, K, L, M, N, P, Q, T, V, W or Y; (76) 226th position: A, D, E, F, G, I, L, M, N, Q, R, S, V, W or Y; (77) 227th position: A, C, E, G, H, I, L, N, P, Q, R, T or V; (78) 230th position: A, C, E, G, K, M, N, P, R, S, T, V, W or Y; (79) 234th position: A, C, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; (80) 236th position: A, C, D, E, F, G, H, K, M, P, R, S, T, V or Y; (81) 239th position: C, D, E, F, G, H, L, M, N, P, Q, R, S, T, W or Y; (82) 242nd position: A, C, D, E, F, G, I, L, M, N, P, Q, R, T, V or Y; (83) 243rd position: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or Y; (84) 244th position: A, C, D, E, F, G, H, I, K, M, Q, R, S, T, V, W or Y; (85) 245th position: C, D, E, F, G, H, L, M, N, P, Q, R, S, V, W or Y; (86) 246th position: A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (87) 247th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W; (88) 248th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, W or Y; (89) 249th position: A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V or W; (90) 250th position: A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; (91) 252nd position: A, C, D, G, H, I, K, L, M, Q, R, S, V or Y; (92) 259th position: A, D, F, G, H, I, L, M, N, P, Q, R, T, W or Y; (93) 261st position: C, D, G, H, I, K, L, P, R, S, T, V, W or Y; (94) 269th position: A, C, D, E, H, I, L, M, P, Q, R, T, V, W or Y; (95) 272nd position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, T, V or Y; (96) 274th position: A, C, D, E, F, H, I, K, L, M, N, P, R, S, T, V or W; (97) 275th position: A, C, D, E, F, I, K, L, M, N, S, T, V, W or Y; (98) 276th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (99) 277th position: A, C, E, G, H, I, K, L, N, Q, R, S, T, V, W or Y; (100) 278th position: A, C, D, E, F, H, I, L, P, Q, S, T, V, W or Y; (101) 279th position: A, C, D, E, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; (102) 280th position: A, D, F, G, H, K, N, P, R, T, W or Y; (103) 281st position: A, C, D, E, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; (104) 291st position: A, C, E, H, I, K, L, P, Q, R, W or Y; (105) 292nd position: A, C, F, G, H, I, L, M, Q, V or W; (106) 307th position: A, C, D, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; (107) 310th position: C, D, E, G, L, M, N, P, Q, R, S, T, V, W or Y; (108) 316th position: A, D, I, L, M, N, V, W or Y; (109) 321st position: A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V or Y; (110) 326th position: A, C, D, F, G, H, L, M, N, P, Q, R, S, V or W; (111) 327th position: A, C, D, F, G, H, K, L, M, N, P, S, T or Y; (112) 328th position: A, D, F, G, I, K, L, N, P, R, T, V, W or Y; (113) 329th position: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, W or Y; (114) 330th position: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (115) 331st position: A, C, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (116) 332nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; (117) 334th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, V or W; (118) 341st position: A, C, E, F, G, K, L, M, N, Q, R, T, V, W or Y; (119) 344th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; (120) 349th position: C, E, G, H, I, M, Q, S, T, V, W or Y; (121) 359th position: A, C, D, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; (122) 364th position: A, C, D, F, H, I, K, L, M, N, P, Q, R, S, V, W or Y; (123) 368th position: A, C, D, E, H, I, K, L, M, N, Q, R, S, V, W or Y; (124) 370th position: A, C, D, G, I, K, L, M, P, Q, R, S, T, V, W or Y; (125) 377th position: D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W; (126) 379th position: C, D, F, H, I, K, L, M, N, P, R, S, T, V or W; (127) 380th position: A, C, D, G, I, K, L, M, P, Q, R, S, T, W or Y; (128) 381st position: A, E, F, G, I, K, L, M, N, P, Q, R, S, V, W or Y; (129) 382nd position: C, D, E, F, G, I, K, L, M, N, Q, R, S, T, V, W or Y; (130) 383rd position: C, D, E, F, G, K, L, M, N, P, Q, R, S, T, W or Y; (131) 389th position: A, D, E, F, G, H, I, K, L, M, N, P, Q, T, V or W; (132) 396th position: A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (133) 401st position: A, D, G, I, L, M, P, Q, R, S, T, V, W or Y; (134) 405th position: C, E, F, H, I, L, M, R, T, V, W or Y; (135) 406th position: A, D, F, G, H, I, K, L, M, P, Q, R, S, T, W or Y; (136) 426th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W or Y; (137) 427th position: A, C, D, E, F, H, K, L, M, N, P, Q, R, S, T, W or Y; (138) 430th position: A, D, E, F, I, K, L, N, P, Q, R, S, V, W or Y; (139) 431st position: D, E, F, G, H, I, K, L, M, N, Q, R, T, V or Y; (140) 432nd position: A, C, D, G, H, I, K, L, M, N, P, R or Y; (141) 433rd position: C, D, E, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y; (142) 434th position: A, C, D, E, F, G, H, K, L, M, P, Q, R, S, T, V, W or Y; (143) 435th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (144) 436th position: C, D, E, G, H, I, K, L, M, N, Q, R, S, V, W or Y; (145) 437th position: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V or W; (146) 438th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, V, W or Y; (147) 439th position: A, C, D, E, F, G, H, K, L, M, N, P, Q, S, V, W or Y; (148) 442nd position: A, C, E, F, G, H, I, K, L, M, N, P, R, S, T, W or Y; (149) 448th position: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (150) 455th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; (151) 457th position: A, C, D, E, F, H, I, L, P, Q, R, S, V, W or Y; (152) 459th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (153) 465th position: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; (154) 466th position: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; (155) 470th position: A, D, E, F, H, I, K, L, M, N, S, T, V or W; (156) 473rd position: F, I, K, L, M, P, R or S; (157) 477th position: A, C, D, E, G, L, M, R, S or Y; (158) 480th position: A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; (159) 481st position: A, C, D, E, F, G, H, I, N, P, Q, R, S, T, V, W or Y; (160) 488th position: A, C, D, E, F, H, I, K, L, M, P, S, T, W or Y; (161) 490th position: C, D, E, G, H, I, K, L, M, N, Q, R, T, V, W or Y; (162) 491st position: A, C, D, E, F, H, K, L, M, N, P, Q, R, S, T, V, W or Y; (163) 492nd position: A, C, D, E, F, G, I, L, M, N, P, Q, R, S, T, V, W or Y; (164) 497th position: A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; (165) 501st position: C, D, E, F, G, I, K, L, M, P, Q, R, S, T, V, W or Y; (166) 504th position: A, D, E, H, I, K, M, N, P, Q, S, T or V; (167) 505th position: A, C, D, E, G, I, K, L, N, P, R, T, V, W or Y; (168) 506th position: A, C, D, F, G, H, K, L, M, N, Q, R, S, T or W; (169) 507th position: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; (170) 522nd position: A, D, E, F, G, H, I, K, L, M, P, Q, S, T, W or Y; (171) 532nd position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V or Y; (172) 537th position: A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y.

4. The monooxygenase mutant as described in claim 3, characterized in that, The activity is the catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II or compound III as shown in structure III.

5. The monooxygenase mutant as described in claim 4, characterized in that, The amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more differences selected from the following amino acid residues:

6. The monooxygenase mutant as described in claim 5, characterized in that, The monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II; Preferably, the amino acid sequence of the monooxygenase mutant differs from that shown in SEQ ID NO:1 by the following amino acid residue differences: 055I / S / T / V, 111C / N / Y, 143D / F / P / R, 210E / H / Q / R / S, 246G / L / M / P, 276D / S / W, 435A / C / I / S / T, 438A / H / M / Y, 448G / L / R, 459D / I / K / Q, and 537F / I / N / S; More preferably, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further includes one or more differences selected from the following amino acid residues: 004H, 006N / T, 045C / V, 059I / L / M / Y, 082A / C / P / S, 083S, 096L / Y, 131I, 165K / Q, 188I, 193V, 214G, 223M / P, 250G, 307H, 330E / Q, 368S, 370G, 380G / K, 381W, 396N / V / Y, 433H / S / V, 455E / N / P / T, 480C / D / V, and 501I / K / V / W; Preferably, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further includes the following combinations of amino acid residue differences: 433H / S / V, 455E / N / P / T, 480C / D / V, and 501I / K / V / W, and one or more selected from the following amino acid residue differences: 004H, 006N / T, 045C / V, 059I / L / M / Y, 082A / C / P / S, 083S, 096L / Y, 131I, 165K / Q, 188I, 193V, 214G, 223P, 250G, 307H, 330E / Q, 368S, 370G, 380G / K, 381W, and 396V / Y.

7. The monooxygenase mutant as described in claim 6, characterized in that, The amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more differences selected from the following amino acid residues: Preferably, the monooxygenase mutant has the activity of catalytically oxidizing compound I as shown in structure I to compound II as shown in structure II, and the yield of compound II as shown in structure II is greater than 90%.

8. The monooxygenase mutant as described in claim 4, characterized in that, The amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more differences selected from the following amino acid residues:

9. The monooxygenase mutant as described in claim 8, characterized in that, The monooxygenase mutant has the activity of catalytic oxidation of compound I as shown in structure I to obtain compound III as shown in structure III; Preferably, the amino acid sequence of the monooxygenase mutant differs from that shown in SEQ ID NO:1 by the following amino acid residue differences: 191C / D / F / G / H / I / K / P / S / W / Y, 329C / I / K / M / R / S / T / V / W / Y, 332C / E / F / H / I / L / M / N / Q / T, 344D / E / F / H / K / M / N / R / T / W, 426D / E / I / N / P / Q / R / S / T / V / W / Y, and 532A / D / E / G / K / P / Q / W / Y; More preferably, the amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, further comprises one or more differences selected from the following amino acid residues: 004H, 006T, 016I / L / N / W / Y, 030D / G / H / Q / R, 055I / K / T / W, 067E / F / I / M / N / P / S / V, 070D / G / K / N / Q / S / T, 083C / D / E / F / I / K / R / S / T, 096L / Y, 111A / D / Y, 141H / P / Q / V / W / Y, 143F / P / R / V, 165K / Q, 185A / C / E / F / H / I, 223M / P, 227E / L / Q / V, 246G, 250G, 259A / C / E / G / R / V, 276D / N, 307H, 368S, 370G, 380G / K, 381A / C / G / K / M / T / V / Y, 435C / F, 438H / Y, 442H / I / S / Y, 448A / R, 459D / I, 497A / C / M / Q / R / V, 501I / K / Q / R / V, 504A / F / H / N / S / T / V, 506E / I / Q / R / S, 522A / F / G / I / R / S / Y and 537F / L / N.

10. The monooxygenase mutant as described in claim 9, characterized in that, The amino acid sequence of the monooxygenase mutant, compared with the amino acid sequence shown in SEQ ID NO:1, contains one or more differences selected from the following amino acid residues: Preferably, the monooxygenase mutant has the activity of catalytically oxidizing compound I as shown in structure I to compound III as shown in structure III, and the yield of compound III as shown in structure III is greater than 90%.

11. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the monooxygenase mutant as described in any one of claims 1 to 10.

12. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 11.

13. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 11 or the recombinant expression vector as described in claim 12; Preferably, the host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells, with Escherichia coli cells being the most preferred.

14. A method for preparing a monooxygenase mutant, characterized in that, The method includes: culturing the transformant as described in claim 13 to obtain the monooxygenase mutant.

15. An enzyme preparation, characterized in that, The enzyme preparation contains the monooxygenase mutant as described in any one of claims 1 to 10; Preferably, the enzyme preparation is a liquid preparation or a lyophilized powder.

16. An enzyme composition, characterized in that, The enzyme composition comprises Bayer Willig monooxygenase or a monooxygenase mutant as described in any one of claims 1 to 10, and one or more of coenzymes, oxidoreductases and catalases. Preferably, the Bayer Willig monooxygenase is derived from Acinetobacter johnsonii, and its amino acid sequence is preferably shown in SEQ ID NO:

1.

17. The use of a Bayer Welig monooxygenase, a monooxygenase mutant as described in any one of claims 1 to 10, an isolated nucleic acid as described in claim 11, a recombinant expression vector as described in claim 12, a transformant as described in claim 13, an enzyme preparation as described in claim 15, and / or an enzyme composition as described in claim 16 in the catalytic oxidation of compound I as shown in structure I to obtain compound II as shown in structure II or compound III as shown in structure III; Preferably, the Bayer Willig monooxygenase is derived from Acinetobacter johnsonii, and its amino acid sequence is preferably shown in SEQ ID NO:

1.

18. A method for preparing compound II as shown in structure II or compound III as shown in structure III, characterized in that, The method comprises: contacting and reacting Bayer Welig monooxygenase, a monooxygenase mutant as described in any one of claims 1 to 10, an enzyme preparation as described in claim 15, and / or an enzyme composition as described in claim 16 with compound I as shown in structure I below to obtain compound II or compound III; 19. The method as described in claim 18, characterized in that, The method includes the following steps: in the presence of an organic solvent, a buffer solution, an oxidant, and catalase, the Bayer Willigan monooxygenase or the monooxygenase mutant is contacted with and reacted with compound I to obtain compound II or compound III; the Bayer Willigan monooxygenase is preferably derived from Acinetobacter johnsonii, and its amino acid sequence is, for example, shown in SEQ ID NO:

1.

20. The method as described in claim 19, characterized in that, The reaction also includes the use of a coenzyme, which includes oxidized coenzyme and reduced coenzyme; Preferably, the reaction further includes the step of regenerating the oxidized coenzyme into the reduced coenzyme; More preferably, the reduced coenzyme is any one or more of NADH and NADPH, and the oxidized coenzyme is NADPH. + and NADP + Any one or more of the following; More preferably, the oxidized coenzyme is regenerated into a reduced coenzyme using NAD. + NADP + NAD + and NADP + Any one of them; More preferably, the reaction further includes a hydrogen donor and an oxidoreductase; preferably, the hydrogen donor is isopropanol, and / or the oxidoreductase is KRED, such as KRED derived from Lactobacillus kefir DSM 20587.

21. The method as described in claim 20, characterized in that, The conditions for the method are selected from one or more of the following: (1) The oxidant is air, a mixture of oxygen and nitrogen, a mixture of oxygen and argon, or a mixture of oxygen and helium; (2) The organic solvent is selected from one or more of ether solvents, epoxy solvents, ester solvents, sulfoxide solvents, amide solvents and alcohol solvents; the sulfoxide solvent is, for example, dimethyl sulfoxide; the alcohol solvent is, for example, selected from one or more of methanol, ethanol, isopropanol and n-butanol; Preferably, the organic solvent is dimethyl sulfoxide; (3) The buffer solution is selected from one or more of sodium tetraborate buffer, phosphate buffer, triethanolamine buffer and tris(hydroxymethyl)aminomethane hydrochloride buffer; (4) The concentration of the buffer solution is 40-60 mM; preferably, the concentration of the buffer solution is 50 mM. (5) The pH value of the buffer solution is 6.0 to 10.5, preferably 7.0 to 10.0, for example 7.0, 7.5, 8.0 or 8.5; (6) The volume-to-mass ratio of the buffer solution to compound I is (10-200) mL / g, preferably (30-100) mL / g; (7) The mass ratio of the Bayer Willig monooxygenase or the monooxygenase mutant to the compound I is (0.01-2):1, preferably (0.05-0.5):1, for example 0.1:1 or 0.2:1; (8) The volume-to-mass ratio of the organic solvent to compound I is (1-50) mL / g, preferably (5-20) mL / g; (9) The mass ratio of the catalase to the compound I is (0.001-0.1):1, preferably (0.01-0.05):1; (10) The mass ratio of the coenzyme to compound I is (0.001 to 0.1):1, preferably (0.01 to 0.05):1; (11) The volume-to-mass ratio of the hydrogen donor to compound I is (1-10) mL / g, preferably 2 mL / g; (12) The mass ratio of the oxidoreductase to compound I is (0.1-1):1; (13) The reaction temperature is 20 to 40°C, preferably 20°C, 30°C or 40°C; (14) The reaction time is 16 to 48 hours, preferably 18 hours.

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