CN115044566B

CN115044566B - Cyclohexanone monooxygenase mutant and application thereof

Info

Publication number: CN115044566B
Application number: CN202210752640.2A
Authority: CN
Inventors: 许国超; 魏世誉; 倪晔
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2023-05-02
Anticipated expiration: 2042-06-29
Also published as: CN115044566A

Abstract

The invention discloses a cyclohexanone monooxygenase mutant and application thereof, belonging to the technical fields of enzyme engineering and microbial engineering. The cyclohexanone monooxygenase mutant has higher activity on various potential chiral sulfides, especially on omeprazole sulfide, under the condition that the wild cyclohexanone monooxygenase can not convert the substrate to produce chiral omeprazole, the mutant can asymmetrically oxidize the potential chiral omeprazole sulfide to produce chiral omeprazole, has higher catalytic efficiency, has higher ee value up to 99%, has higher conversion efficiency on other potential chiral sulfides, and has good industrial application prospect.

Description

Cyclohexanone monooxygenase mutant and application thereof

Technical Field

The invention relates to a cyclohexanone monooxygenase mutant and application thereof, belonging to the technical fields of enzyme engineering and microbial engineering.

Background

Chiral sulfoxide is an important chiral compound, can be used for synthesizing chiral auxiliary reagents and chiral ligands, and has wide application in the field of medicines.

Esomeprazole (S-omeprazole), chemically named (S) -5-methoxy-2- [ [ (4-methoxy-3, 5-dimethyl-2-pyridinyl) methyl ] sulfinyl ] -1H-benzimidazole, is a Proton Pump Inhibitor (PPI) designated for the treatment of dyspepsia, peptic Ulcer (PUD), gastroesophageal reflux disease (GORD/GERD) and Zollinger-Ellison syndrome, and is also the first optically pure proton pump inhibitor on the market. Esomeprazole has better acid inhibiting effect than the raceme omeprazole. The production method of esomeprazole mainly comprises a chemical synthesis method and a biological enzyme method, the existing mature chemical synthesis method has complex reaction and more byproducts, and the biological method has important research value due to environmental protection, simple steps and high stereoselectivity. The biological catalysis method for producing the chiral medicine esomeprazole has huge market space in the pharmaceutical industry. At present, chemical methods are mainly adopted at home and abroad to produce the chiral medicine esomeprazole, and biological methods for producing the esomeprazole are still in a laboratory research stage. The foreign research on the biological enzyme catalysis method for producing esomeprazole is more and a certain research result is obtained, and the domestic research on the aspect still has a big gap.

In 2013, the American Codexis company uses an engineered cyclohexanone monooxygenase mutant with more than 30 mutation sites, 100g/L omeprazole thioether substrate is added into a 75mL reaction system, the mixture is degassed and aerated for 4 times, fed-batch enzyme and coenzyme are reacted in an oxygen atmosphere under stirring, the rotation speed is 200rpm, the total conversion is 69 hours, and finally, the conversion rate reaches 99.6%, and the e.e. value is 99.9%. In 2011, peter, eva, etc., isolated a strain of lysine bacillus that oxidized omeprazole sulfide. The method adopts a fed-batch enzyme and substrate mode, the amount of added thalli is 25mg CDW in every 5mL of LB culture medium, the total addition amount of the substrate is 0.1g/L, and the substrate is transformed for 24 hours by shaking at 200rpm, so that the 77% conversion rate is finally obtained, and the e.e. value is 100%. In 2019, xu Jian and team constructed an engineered cyclohexanone monooxygenase mutant with more than 15 mutation sites, selectively oxidizing omeprazole sulfide to produce the desired (S) -omeprazole. Omeprazole thioether was completely converted within 6h when loaded with 15mM substrate. The CHMO has great application potential in the field of chiral drug synthesis, wherein the patent of the optically pure esomeprazole (including sodium salt, magnesium salt, lithium salt, potassium salt, calcium salt and the like) critical in 5 months in 2014 is expired, the market with abundant profits is faced, but the chemical process is complex, the environment pollution is caused, the byproducts are more, the optical selectivity is not high and the like, and the biological method for producing the esomeprazole has the advantages of environmental friendliness, mild conditions, strong characteristics and the like, and is receiving more and more attention. Cyclohexanone monooxygenase has the potential for industrial production due to its high stereoselectivity for omeprazole thioether.

Therefore, there is an urgent need to obtain more cyclohexanone monooxygenases for producing chiral esomeprazole by asymmetrically oxidizing latent chiral omeprazole sulfide so as to realize large-scale industrial production of chiral esomeprazole in the early days and large-scale application of chiral esomeprazole in the medicine field.

Disclosure of Invention

In order to solve the problems, the invention provides cyclohexanone monooxygenase (Cyclohexanone monooxygenase, CHMO for short, EC 1.1.1.1) with high conversion efficiency for producing chiral sulfoxide by asymmetrically oxidizing latent chiral thioether.

The first object of the invention is to provide a cyclohexanone monooxygenase mutant, which is obtained by mutating 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine (F434I) and mutating 435 th threonine into alanine (T435A) to obtain mutant F434I/T435A.

Further, the mutant is obtained by mutating 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 into isoleucine (F434I), mutating 435 th threonine into alanine (T435A), and mutating 437 th leucine into cysteine (L437C), thereby obtaining mutant F434I/T435A/L437C.

Further, the mutant is obtained by mutating 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 into isoleucine (F434I), mutating 435 th threonine into alanine (T435A), mutating 437 th leucine into cysteine (L437C), and mutating 507 th phenylalanine into valine (F507V), thus obtaining mutant F434I/T435A/L437C/F507V.

Further, the mutant is characterized in that 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 is mutated into isoleucine (F434I), 435 th threonine is mutated into alanine (T435A), 437 th leucine is mutated into cysteine (L437C), 507 th phenylalanine is mutated into valine (F507V), 428 th leucine is mutated into glutamine (L428Q), and thus the mutant F434I/T435A/L437C/F507V/L428Q is obtained.

Further, the mutant is characterized in that 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 is mutated into isoleucine (F434I), 435 th threonine is mutated into alanine (T435A), 437 th leucine is mutated into cysteine (L437C), 507 th phenylalanine is mutated into valine (F507V), 428 th leucine is mutated into glutamine (L428Q), and 440 th serine is mutated into isoleucine (S440I), so that the mutant F434I/T435A/L437C/F507V/L428Q/S440I is obtained.

Further, the mutant is characterized in that 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 is mutated into isoleucine (F434I), 435 th threonine is mutated into alanine (T435A), 437 th leucine is mutated into cysteine (L437C), 507 th phenylalanine is mutated into valine (F507V), 428 th leucine is mutated into glutamine (L428Q), 440 th serine is mutated into isoleucine (S440I), and 328 th lysine is mutated into cysteine (K328C), so that the mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C is obtained.

Further, the mutant is characterized in that 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 is mutated into isoleucine (F434I), 435 th threonine is mutated into alanine (T435A), 437 th leucine is mutated into cysteine (L437C), 507 th phenylalanine is mutated into valine (F507V), 428 th leucine is mutated into glutamine (L428Q), 440 th serine is mutated into isoleucine (S440I), 328 th lysine is mutated into cysteine (K328C), and meanwhile, 435 th phenylalanine is mutated into valine (F279V), so that the mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V is obtained.

Further, the mutant is characterized in that the 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown in SEQ ID NO.1 is mutated into isoleucine (F434I), the 435 th threonine is mutated into alanine (T435A), the 437 th leucine is mutated into cysteine (L437C), the 507 th phenylalanine is mutated into valine (F507V), the 428 th leucine is mutated into glutamine (L428Q), the 440 th serine is mutated into isoleucine (S440I), the 328 th lysine is mutated into cysteine (K328C), the 279 th phenylalanine is mutated into valine (F279V), and the 290 th alanine is mutated into valine (A290V), so that the mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V/A290V is obtained.

Further, the mutant is characterized in that the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine (F434I), the 435 th threonine is mutated to alanine (T435A), the 437 th leucine is mutated to cysteine (L437C), the 507 th phenylalanine is mutated to valine (F507V), the 428 th leucine is mutated to glutamine (L428Q), the 440 th serine is mutated to isoleucine (S440I), the 328 th lysine is mutated to cysteine (K328C), the 279 th phenylalanine is mutated to valine (F279V), the 290 th alanine is mutated to valine (A290V), the 245 th valine is mutated to alanine (V245A), and the 246 th valine is mutated to leucine (V246L), so that F434I/T A/L437C/F507I/S440I/K328V/F290V 245V/V246L mutant is obtained.

Further, the mutant is characterized in that the 434 th phenylalanine of cyclohexanone monooxygenase with the amino acid sequence shown in SEQ ID NO.1 is mutated to isoleucine (F434I), the 435 th threonine is mutated to alanine (T435A), the 437 th leucine is mutated to cysteine (L437C), the 507 th phenylalanine is mutated to valine (F507V), the 428 th leucine is mutated to glutamine (L428Q), the 440 th serine is mutated to isoleucine (S440I), the 328 th lysine is mutated to cysteine (K328C), the 245 th phenylalanine is mutated to valine (F279V), the 290 th alanine is mutated to valine (A290V), the 245 th valine is mutated to alanine (V245A), the 246 th valine is mutated to leucine (V246L), the 491 th serine is mutated to cysteine (S492C), and meanwhile the 492 th leucine is mutated to arginine (W507R), so that the mutant F I/T435A/L/F507/F/V428V/F492V/F434A/V/F290L is obtained.

It is a second object of the present invention to provide a gene encoding the cyclohexanone monooxygenase mutant.

A third object of the present invention is to provide a recombinant plasmid carrying the gene.

Further, the recombinant plasmid vector is pET-28a (+) plasmid, pET-28b (+) plasmid or pET-20b (+) plasmid.

It is a fourth object of the present invention to provide host cells expressing the cyclohexanone monooxygenase mutants.

Further, the host cell is a bacterial, fungal, plant cell or animal cell.

Further, the bacterium is E.coli, preferably E.coli BL21 (DE 3).

Further, the cyclohexanone monooxygenase mutant is prepared by the following steps: inoculating the host cell expressing the cyclohexanone monooxygenase mutant into a fermentation medium for fermentation to obtain fermentation liquor; centrifuging the fermentation liquor, and collecting thalli; crushing thalli, and centrifuging to obtain cell crushing supernatant; and extracting cell disruption supernatant to obtain the cyclohexanone monooxygenase mutant.

It is a fifth object of the present invention to provide the use of the above cyclohexanone monooxygenase mutant, gene, expression vector or host cell in the production of chiral sulphoxide.

Further, the application is that the cyclohexanone monooxygenase mutant is added into a reaction system containing the potential chiral thioether for reaction to obtain a reaction solution; and extracting the reaction liquid to obtain chiral sulfoxide.

Further, the latent chiral thioether is phenyl sulfide, 2-methylthionaphthalene, benzyl phenyl sulfide or omeprazole sulfide.

Further, the chiral sulfoxide is benzyl sulfoxide, 2- (methylsulfinyl) naphthalene, benzyl phenyl sulfoxide or chiral omeprazole. Specifically, when the chiral phenyl sulfide is latent, the chiral sulfoxide is phenyl sulfoxide; when the potential chiral 2-methylthionaphthalene is, the chiral sulfoxide is 2- (methylsulfinyl) naphthalene; when the latent chiral benzyl phenyl sulfide is adopted, the chiral sulfoxide is benzyl phenyl sulfoxide; when the potential chiral omeprazole thioether is adopted, the chiral sulfoxide is chiral omeprazole.

Further, the chiral sulfoxide is (S) -omeprazole or (R) -omeprazole.

Further, the reaction system containing the latent chiral thioether also contains coenzyme and a coenzyme circulation system; the coenzyme circulation system contains D-glucose and glucose dehydrogenase, or contains formate and formate dehydrogenase, or contains lactate and lactate dehydrogenase, or contains glycerol and glycerol dehydrogenase.

Further, the coenzyme is NADP ⁺ 、NADPH、NAD ⁺ And NADH.

Further, the addition amount of the cyclohexanone monooxygenase mutant in a reaction system is 1-10 kU/L.

Further, the concentration of the latent chiral thioether in the reaction system is 1-5 mmol/L.

Further, the concentration of the coenzyme in the reaction system is 0.1 to 1mmol/L.

Further, the concentration of glucose dehydrogenase in the reaction system is 1 to 10kU/L.

Further, the concentration of D-glucose in the reaction system is 2-10 mmol/L.

Further, the reaction system is a buffer solution containing the potential chiral thioether, the coenzyme and a coenzyme circulation system.

Further, the buffer solution is Tris-HCl buffer solution.

Further, the concentration of the Tris-HCl buffer solution is 0.1-0.2 mol/L.

Further, the reaction temperature is 30-35 ℃ and the pH is 7-9.

The invention has the beneficial effects that:

(1) The cyclohexanone monooxygenase mutant disclosed by the invention has high conversion efficiency in producing chiral sulfoxide by asymmetrically oxidizing latent chiral thioether, wherein the catalytic efficiency in producing chiral (R) -omeprazole by using mutant M2 asymmetrically oxidizing latent chiral omeprazole thioether is 0.144min ^–1 ·mM ^–1 The ee value can reach 99% R. And the wild type cannot convert the omeprazole thioether to produce (R) -omeprazole under the same conditions.

(2) The catalytic efficiency of the cyclohexanone monooxygenase mutant for producing chiral sulfoxide is high, wherein the catalytic efficiency of the mutant M3 for producing chiral (R) -omeprazole by asymmetrically oxidizing latent chiral omeprazole thioether is 5.83min ^–1 ·mM ^–1 The ee value is 40 times of that of the mutant M2, and can reach 99% R.

(3) The catalytic efficiency of the cyclohexanone monooxygenase mutant for producing chiral sulfoxide is higher, wherein the catalytic efficiency of the mutant M7 for producing chiral (S) -omeprazole by asymmetrically oxidizing latent chiral omeprazole thioether is 0.06min ^–1 ·mM ^–1 The ee value can reach 97.3 percent S. And the wild type cannot convert the omeprazole thioether to produce (S) -omeprazole under the same conditions.

(4) The catalytic efficiency of the cyclohexanone monooxygenase mutant for producing chiral sulfoxide is higher, wherein the catalytic efficiency of the mutant M10 for producing chiral (S) -omeprazole by asymmetrically oxidizing latent chiral omeprazole thioether is 0.38min ^–1 ·mM ^–1 The ee value of the mutant M7 is 6.3 times of that of the mutant M7, and can reach 97.3 percent S.

(5) The wild cyclohexanone monooxygenase can asymmetrically oxidize the potential chiral 2-methylthionaphthalene and the benzyl phenyl thioether, and the ee value can reach 99 percent (R) and 99 percent (S) respectively; the cyclohexanone monooxygenase mutant can asymmetrically oxidize the latent chiral 2-methylthionaphthalene and the benzyl phenyl thioether, wherein the mutant M2-M6 can asymmetrically oxidize the latent chiral 2-methylthionaphthalene and the benzyl phenyl thioether, and the ee value can reach 99% (R) and 99% (S) respectively. Both viability and selectivity remained consistent with wild type.

(6) The catalytic efficiency of the cyclohexanone monooxygenase mutant for producing chiral sulfoxides by asymmetrically oxidizing latent chiral sulfoether is high, so that the cyclohexanone monooxygenase mutant has extremely high application prospect in producing chiral sulfoxides such as (S) -omeprazole, (R) -omeprazole and the like.

Drawings

FIG. 1 is a PCR amplification electropherogram for recombinant plasmids; wherein M: marker, lanes 1-11: PCR products of wild type WT and mutant recombinant plasmids pET28 a-AmCHMO-1-recombinant plasmids pET28a-AmCHMO-10.

FIG. 2 shows the result of SDS-PAGE analysis of the expression product obtained by shake flask induced fermentation of recombinant E.coli; wherein M: standard protein Maker, lanes 1-11: wild type and mutant recombinant escherichia coli E.coli BL21/pET28 a-AmCHMO-1-recombinant escherichia coli E.coli BL21/pET28a-AmCHMO-10 shake flask induced fermentation to obtain pure enzyme of wild type WT and mutant M1-M10.

Figure 3 is a chiral chromatogram of (R) -omeprazole and (S) -omeprazole obtained as a result of asymmetric oxidation of latent chiral omeprazole sulfide by mutants M3 and M10.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

Coli e.coli BL21 (DE 3) as referred to in the examples below was purchased from north-nano organisms; the pET-28a (+) plasmid and NADPH referred to in the examples below were purchased from Novagen; glucose Dehydrogenase (GDH), lactate Dehydrogenase (LDH) referred to in the examples below were purchased from Novain; the D-glucose referred to in the examples below was purchased from Jieli corporation; the latent chiral thioethers referred to in the examples below were purchased from the national drug group (E.coli BL21 (DE 3) strain described above is commercially available and does not require preservation for the patent procedure).

The following examples relate to the following media:

LB liquid medium: yeast powder 5.0 g.L ^-1 Tryptone 10.0 g.L ^-1 、NaCl 10.0g·L ^-1 Kanamycin 100 mg.L ^-1 。

LB solid medium: yeast powder 5.0 g.L ^-1 Tryptone 10.0 g.L ^-1 、NaCl 10.0g·L ^-1 15g/L of agar powder and 50 mg.L of kanamycin ^-1 。

The detection method involved in the following examples is as follows:

the detection method of the enzymatic activity of the cyclohexanone monooxygenase comprises the following steps:

after incubating Tris-HCl buffer (100 mM, ph 9.0) containing 1mM NADPH, 1.0mM substrate prochiral omeprazole thioether at 30 ℃ for 2min, 10 μl of pure enzyme solution was added to Tris-HCl buffer to react at 30 ℃ for 30min, and the reaction was stopped with acetonitrile of equal volume, and the amount of product produced was measured by high performance liquid chromatography, based on which the enzyme activity was calculated;

the calculation formula of the enzyme activity is as follows:

enzyme activity (u/mg) = (c1×v1) ×10 ³ /(T×C2×V2)；

Wherein, the concentration of the C1 generated product is expressed as M/L; v1 is the volume of the reaction solution, and the unit is mL; c2 is the protein concentration of the enzyme in mg/ml; v2 is the volume of enzyme added in the reaction system, and the unit is mu L; t is the reaction time in min;

definition of enzyme activity: the amount of enzyme required to catalytically oxidize l. Mu. Mol of substrate per minute to produce l. Mu. Mol of product under this condition is one enzyme activity unit (1 u).

The method for detecting the conversion efficiency and stereoselectivity of the cyclohexanone monooxygenase asymmetric oxidation potential chiral omeprazole thioether to chiral (S) -omeprazole and (R) -omeprazole comprises the following steps:

after incubating Tris-HCl buffer (100 mM, pH 9.0) containing 1mM NADPH and 1.0mM substrate prochiral omeprazole thioether at 30℃for 2min, 10. Mu.L of pure enzyme solution was added to Tris-HCl buffer and reacted at 30℃for 60min; adding 500 mu L of ethyl acetate after the reaction is finished, oscillating for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifuge tube, adding 500 mu L of chromatographic pure ethanol until the organic phase is completely volatilized naturally, and carrying out chiral liquid chromatography analysis on conversion efficiency and ee value;

the conversion efficiency chromatographic conditions are specifically as follows: c18 (5 μm,250mm×4.6mm) liquid chromatographic column, acetonitrile as mobile phase, water (53:47, v/v/v), flow rate 1mL/min, column temperature 30 ℃, ultraviolet detection wavelength 254nm, sample injection amount 10 μl, and retention time of substrate omeprazole thioether and product omeprazole 4.12min and 8.71min respectively;

the stereoselective chromatography conditions are specifically as follows: daicel Chiralcel AD-3 (5 μm,250 mm. Times.4.6 mm) liquid chromatographic column with normal hexane-isopropanol acetic acid (50:50:0.01, v/v/v), flow rate of 0.5mL/min, column temperature of 30deg.C, ultraviolet detection wavelength of 250nm, sample injection amount of 10 μl, and retention time of (S) -omeprazole and (R) -omeprazole of 11.12min and 13.71min respectively;

the conversion efficiency was calculated as follows:

the ee value is calculated as follows:

as: the molar concentration of (S) -omeprazole in the reaction solution; a is that _R : the molar concentration of (R) -omeprazole in the reaction solution; a is that _sub : the molar concentration of unreacted omeprazole thioether in the reaction solution.

EXAMPLE 1 construction, expression and purification of Cyclohexanone monooxygenase mutant

Chemically synthesizing a gene of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 (the nucleotide sequence of the gene is shown as SEQ ID NO. 2); connecting the obtained gene with pET-28a (+) plasmid after double digestion (NdeI and XhoI), converting the connection product into E.coliBL21 (DE 3), coating the conversion product on LB solid medium, culturing for 8-10 h at 37 ℃, picking up 5 transformants on LB solid medium, inoculating LB liquid medium for culturing, extracting plasmid after culturing for 10h at 37 ℃, carrying out enzyme digestion verification and sequencing verification on the extracted plasmid, and obtaining recombinant plasmid pET28a-AmCHMO containing the gene encoding wild-type cyclohexanone monooxygenase and recombinant bacterium E.coliBL 21/pET28a-AmCHMO containing the gene encoding wild-type cyclohexanone monooxygenase after verification.

Performing site-directed mutagenesis by using the obtained recombinant plasmid pET28a-AmCHMO as a template to obtain a plasmid containing a coding cyclohexanone monooxygenase mutant F434I (the 434 th phenylalanine is mutated to isoleucine), T435A (the 435 th threonine is mutated to alanine), L437C (the 437 th leucine is mutated to cysteine), F507V (the 507 th phenylalanine is mutated to valine), L428Q (the 428 th leucine is mutated to glutamine), S440I (the 440 th serine is mutated to isoleucine), K328C (the 328 th lysine is mutated to cysteine), F279V (the 279 th phenylalanine is mutated to valine), A290V (the 290 th alanine is mutated to valine), V245A/V246L (the two valines at 246 th are mutated to alanine and leucine respectively), S491C/W492R (the 491 th serine and tryptophan are mutated to cysteine and arginine);

mutation site and number of mutants: F434I/T435A (M1), F434I/T435A/L437C (M2), F434I/T435A/L437C/F507V (M3), F434I/T435A/L437C/F507V/L428Q/S440I (M4), F434I/T435A/L437C/F507V/L428Q/S440I (M5), F434I/T435A/L437C/F507V/L428Q/S440I/K328C (M6), F434I/T435A/L437C/F507V/L428Q/L440I/K328C/F279V (M7), F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V (M8), F434I/T435A/L507V/L507Q/L440I/K328C/K328V/F328C/F328V (M5), F434I/L435A/L437C/L428V/F328V/L428V (M35A/L245V) and F245V (M245A 245V) 35C/L245V (M10A/L245V) or F245V 279/F245V (M245) 35F 245I/F245V/L245V/F245, the cyclohexanone monooxygenase mutants are respectively named as M1-M10;

wherein, the primers used for the mutations F434I, T435A, L437C, F507V, L428Q, S I, K328C, F279V, A290V, V A/V246L, S491C/W492R are as follows:

F434I-F：CCTAACGGCCCGatcGCTAAT；

F434I-R：CGGGCAATTAGCgatCGGGCC；

T435A-F：AACGGCCCGCTGgctAATTGC；

T435A-R：CGGCGGGCAATTagcCAGCGG；

L437C-F：CCGatcgctAATtgcCCGCCG；

L437C-R：GGCGGAHNATTgcaCAGagc；

F507V-F：AATGCAGTGATGgtgTATCTG；

F507V-R：GCCTGCCAGATAcacCATCAC；

L428Q-F：ATGTTTATGATTCcagGTCCT；

L428Q-R：GCCGTTAGGACCctgAATCAT；

S440I-F：TGCtgcCCGATCatcGAAACC；

S440I-R：GGTTTCAATGATgtaCGGGCA；

K328C-F：GATTTATATGCCtgcCGTCCG；

K328C-R：GCACAGCGGACGgcaGGCATA；

F279V-F：AAAGGCAATGGTgtgCGCTTT；

F279V-R：GAACATAAAGCGcacACCATT；

A290V-F：TTTTGTGATATTgtgACCAAT；

A290V-R：TTCCGGATTGGTcacAATATC；

V245A/V246L-F：GTGCGTAATAGTGCTgctctgTTTGGT；

V245A/V246L-R：AAAACCAAAGGCCAcagagcTATTACG；

S491C/W492R-F：CCGAAAGCCGATtgccgaATTTTT；

S491C/W492R-R：TGCACCAAAAATtcggcaATCGGC，

the PCR reaction system (50. Mu.L) was: l.0. Mu.L of KOD enzyme (2.5U/mL), l.0. Mu.L of template (5-50 ng), 4.0. Mu.L of dNTP, 5.0. Mu.L of 10 Xreaction buffer, 1.0. Mu.L of each of the upstream and downstream primers, and up to 50. Mu.L of ddH 2O;

the PCR product amplification conditions were: (1) denaturation at 94℃for 3min, (2) denaturation at 94℃for 30sec, (3) annealing at 54℃for 30sec, (4) extension at 72℃for 150sec, repeating steps (2) to (4) for 10 to 15 cycles, finally extension at 72℃for 10min, and preservation of PCR amplification product at 4 ℃.

Detecting the PCR amplified product by using 1% agarose gel electrophoresis, adding 0.5 mu L of methylation template digestive enzyme (Dpn I) into 10 mu L of amplified product after detection, uniformly mixing by blowing and sucking at a gun head, reacting for 1h at 37 ℃, converting the amplified product treated by Dpn I into escherichia coli E.coli BL21 (DE 3), coating the converted product on LB solid culture medium, culturing for 8-10 h at 37 ℃, picking up 3 transformants on LB solid culture medium, inoculating LB liquid culture medium for culturing for 10h, extracting plasmids after culturing for 37 ℃, carrying out enzyme digestion verification (the verification result can be seen in figure 1) and sequencing verification, and obtaining recombinant plasmids pET28 a-AmCHMO-1-recombinant plasmids pET28a-AmCHMO-10 respectively containing genes for encoding cyclohexanone monooxygenase mutants M1-M10 and recombinant plasmids E.coli BL 28a-Am 28 a-AmCHMO-Am 21-Am 10 respectively.

The obtained recombinant bacteria E.coli BL21/pET28 a-AmCHMO-1-recombinant bacteria E.coli BL21/pET28a-AmCHMO-10 are respectively coated on LB solid culture medium, and are cultured for 8-10 h at 37 ℃ to obtain single colony; selecting single colony, inoculating the single colony into LB liquid culture medium, and culturing for 12-14 h at 37 ℃ to obtain seed liquid; inoculating the seed solution into LB liquid culture medium according to 2% (v/v) inoculum size, culturing at 37deg.C and 200rpm to OD ₆₀₀ After reaching 0.8, adding IPTG with the final concentration of 0.2mM into the fermentation broth, and continuously performing induction culture at 25 ℃ for 8 hours to obtain the fermentation broth; centrifuging the fermentation broth at 4deg.C and 8000rpm for 10min, and collecting cells; the cells thus collected were suspended in potassium phosphate buffer (100 mmol.L) ^-1 Ultrasonic disruption was performed at pH 6.0), and cell disruption supernatants each containing wild-type cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M1 to M10 were collected.

The cell disruption supernatant obtained was purified using an affinity column HisTrap FF credit (nickel column) as follows: buffer A (20 mmol.L) ^-1 Tris-HCl，500mmol·L ^-1 NaCl，20mmol·L ^-1 Imidazole, pH 7.4), and passing the cell disruption supernatant obtained in example 1 through a nickel column, eluting the unbound proteins with buffer A, and after the breakthrough peak has been completed, passing from buffer A to buffer B (20 mmol.L ^-1 Tris-HCl，500mmol·L ^- ¹ NaCl，500mmol·L ^-1 Imidazole, pH 7.4), eluting the recombinant protein bound to the nickel column to obtain pure enzyme solutions of wild cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M1-M10.

The obtained pure enzyme solutions of the cyclohexanone monooxygenase mutants M1 to M10 were subjected to SDS-PAGE analysis, and the analysis results are shown in FIG. 2.

As shown in FIG. 2, the pure enzyme solutions of the cyclohexanone monooxygenase mutants M1 to M10 all showed single bands at about 59kDa, and the impurity proteins were less, indicating that the nickel column purification effect was better.

Example 2 kinetic parameters of Cyclohexanone monooxygenase mutants and stereoselectivity of asymmetric oxidation of latent chiral sulfides to chiral sulfoxides

Selecting the wild-type cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M1-M10 obtained in example 1, respectively taking the potential chiral omeprazole thioether with the concentration of 0.1-1 mM as a substrate, measuring the initial oxidation activity of the wild-type cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M2-M10 obtained in example 1, fitting the data by adopting a nonlinear regression method in Graph Pad Prism 7.0 software to respectively obtain K of Mi (Michaelis-Menten) equation _m The value is recalculated to obtain K _cat And K _cat /K _m Values, calculated results are shown in table 1; wherein K is _cat The calculation formula of the value is: k (K) _cat ＝V _max M/1; wherein M is the molecular mass of the enzyme in kDa.

From Table 1, the catalytic efficiency of the cyclohexanone monooxygenase mutant M3 asymmetric oxidation potential chiral omeprazole thioether to generate (R) -omeprazole is obviously improved compared with that of the wild cyclohexanone monooxygenase; the catalytic efficiency of the cyclohexanone monooxygenase mutant M8 for asymmetrically oxidizing the potential chiral omeprazole thioether to generate (S) -omeprazole is improved by 10 times compared with that of the cyclohexanone monooxygenase mutant M7; the catalytic efficiency of the cyclohexanone monooxygenase mutant M10 for asymmetrically oxidizing the potential chiral omeprazole thioether to generate (S) -omeprazole is improved by 5 times compared with that of the cyclohexanone monooxygenase mutant M8.

The wild type cyclohexanone monooxygenase obtained in example 1 and cyclohexanone monooxygenase mutants M2 to M10 were tested for stereoselectivity and relative activity of chiral sulfoxides produced by asymmetric oxidation of latent chiral sulfoxides, and the test results are shown in Table 2.

As shown in Table 2, the stereoselectivity of the cyclohexanone monooxygenase mutants M2 to M6 for asymmetrically oxidizing the latent chiral phenylsulfide to generate the chiral sulfoxide is not reduced compared with that of the wild cyclohexanone monooxygenase; but the relative activity gradually decreases, and when the cyclohexanone monooxygenase mutants M7-M10 are reached, the activity of the asymmetric oxidation latent chiral phenylsulfide is lost; as shown in Table 2, the wild cyclohexanone monooxygenase can asymmetrically oxidize the latent chiral 2-methylthionaphthalene, and the stereoselectivity of the cyclohexanone monooxygenase mutant M2-M5 for asymmetrically oxidizing the latent chiral phenyl sulfide to generate the chiral sulfoxide is not reduced compared with that of the wild cyclohexanone monooxygenase, the ee value can reach 99% (R), and the activity is basically kept unchanged; compared with the wild cyclohexanone monooxygenase, when the cyclohexanone monooxygenase mutant M6-M10 asymmetrically oxidizes the latent chiral 2-methylthionaphthalene, the selectivity is reduced, and the activity is firstly reduced and then increased; for benzyl phenyl sulfide, the selectivity of cyclohexanone monooxygenase mutants M3 to M6 was not reduced compared to wild-type cyclohexanone monooxygenase, except for cyclohexanone monooxygenase mutant M2; for the omeprazole thioether, the activity of the cyclohexanone monooxygenase mutant M2-M10 for asymmetrically oxidizing the latent chiral omeprazole ether to generate chiral omeprazole is obviously improved compared with that of the wild cyclohexanone monooxygenase; wherein, the activity of the cyclohexanone monooxygenase mutant M2-M3 for asymmetrically oxidizing the potential chiral omeprazole thioether to generate chiral (R) -omeprazole is gradually improved, and the selectivity is kept unchanged. The stereoselectivity of the cyclohexanone monooxygenase mutants M4-M6 for asymmetrically oxidizing the latent chiral omeprazole ether to generate chiral omeprazole is gradually reduced; the relative activity is gradually reduced, and the activity is reduced to the minimum by the cyclohexanone monooxygenase mutant M7, but the selectivity is overturned, the ee value can reach 97.3 percent (S), and then the cyclohexanone monooxygenase mutants M8-M10 keep the selectivity unchanged, but the activity is gradually improved.

TABLE 1 kinetic parameters of wild-type cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M1-M10

Entry	Km(mM)	kcat(min ^–1 )	kcat/Km(min ^–1 ·mM ^–1 )
				WT	ND	ND	ND
M1	0.125	0.006	0.048
				M2	0.292	0.042	0.144
M3	0.247	1.44	5.834
				M4	0.232	0.356	1.534
M5	0.256	0.111	0.435
				M6	0.237	0.015	0.063
M7	0.028	0.002	0.072
				M8	0.157	0.017	0.108
M9	0.153	0.074	0.136
				M10	0.215	0.088	0.380

ND no vitality was measured.

TABLE 2 relative Activity and stereoselectivity of wild type Cyclohexanone monooxygenase and Cyclohexanone monooxygenase mutants M2-M10 for asymmetric Oxidation of latent chiral sulfides to chiral sulfoxides

ND no vitality was measured.

Example 3 conversion efficiency of Cyclohexanone monooxygenase mutant to sulfoxide by asymmetric Oxidation of potential chiral thioether

The cyclohexanone monooxygenase mutants M1-M10 obtained in example 1 were selected, and the cyclohexanone monooxygenase mutants M1-M10 obtained in example 1 were added to 100mM Tris-HCl buffer (pH 9.0) containing 5mM, 2-methylthionaphthalene, and benzyl phenyl sulfide, respectively, in an amount of 2g/L, and reacted at 30℃and pH 9.0 at 200rpm for 1-12 hours to obtain reaction solutions; in addition to the prochiral omeprazole thioether, tris-HCl buffer also contains coenzyme NADP+ in a concentration of 0.02mM, glucose in a concentration of 1.5mM, glucose dehydrogenase GDH in a concentration of 1.5mM and methanol in a concentration of 5% (v/v).

The conversion rate and selectivity of the cyclohexanone monooxygenase mutants M1-M10 to the corresponding sulfoxide by asymmetrically oxidizing the potential chiral phenylsulfide, 2-methylthionaphthalene and benzyl phenyl sulfide at different reaction times are respectively detected, and the detection results are shown in Table 3.

TABLE 3 conversion of Cyclohexanone monooxygenase mutants M1-M10 into the corresponding sulfoxides by asymmetric oxidation of latent chiral phenylsulfide, 2-methylthionaphthalene and benzylphenyl sulfide

ND no vitality was measured.

As can be seen from Table 3, at a substrate concentration of 5mM, the conversion rates of the cyclohexanone monooxygenase mutants M1 to M10 respectively reach >90% in the reaction for 1 to 12 hours, wherein the conversion rates of the cyclohexanone monooxygenase mutants M1 to M3 for asymmetrically oxidizing the latent chiral phenylsulfide, 2-methylthionaphthalene and benzyl phenyl sulfide to the corresponding sulfoxides are consistent with those of the wild type cyclohexanone monooxygenase. The conversion rate of the cyclohexanone monooxygenase mutant M4-M10 asymmetric oxidation potential chiral phenylsulfide, 2-methylthionaphthalene and benzyl phenyl sulfide to generate corresponding sulfoxide is obviously reduced compared with the conversion rate of the wild cyclohexanone monooxygenase. The above results demonstrate that as the mutation site increases, the substrate binding pocket of the mutant also gradually expands and gradually loses catalytic activity for a smaller substrate compared to the wild-type cyclohexanone monooxygenase.

EXAMPLE 4 conversion efficiency of Cyclohexanone monooxygenase mutant to asymmetric Oxidation of latent chiral omeprazole thioether to (R) -omeprazole and (S) -omeprazole

The cyclohexanone monooxygenase mutants M1-M10 obtained in example 1 were selected, the cyclohexanone monooxygenase mutants M1-M10 obtained in example 1 were added to 100mM Tris-HCl buffer (pH 9.0) containing 2mM, potentially chiral omeprazole sulfide, respectively, in an amount of 2g/L, and reacted at 30℃and pH 9.0 at 200rpm for 1-12 hours to obtain reaction solutions; in addition to the prochiral omeprazole thioether, tris-HCl buffer also contains coenzyme NADP+ in a concentration of 0.02mM, glucose in a concentration of 1.5mM, glucose dehydrogenase GDH in a concentration of 1.5mM and methanol in a concentration of 5% (v/v).

The conversion rate and selectivity of the cyclohexanone monooxygenase mutant M1-M10 asymmetric oxidation potential chiral omeprazole thioether to (S) -omeprazole and (R) -omeprazole are respectively detected at different reaction times, and the conversion efficiency of the cyclohexanone monooxygenase mutant M3 and the cyclohexanone monooxygenase mutant M10 asymmetric oxidation potential chiral omeprazole thioether to (R) -omeprazole and (S) -omeprazole are calculated, and the detection results are shown in Table 4.

TABLE 4 conversion of Cyclohexanone monooxygenase mutants M1-M10 asymmetric oxidation of potentially chiral omeprazole thioethers to (R) -omeprazole or (S) -omeprazole

ND no vitality was measured.

As can be seen from Table 4, at a substrate concentration of 2mM, the cyclohexanone monooxygenase mutants M1-M10 respectively reached a conversion rate of >99% in the reaction for 1-12 hours, while the wild-type cyclohexanone monooxygenase was not converted, and it can be seen that the conversion efficiency of the cyclohexanone monooxygenase mutants M1-M10 for asymmetric oxidation of latent chiral omeprazole sulfide to (R) -omeprazole and (S) -omeprazole was significantly improved compared with that of the wild-type cyclohexanone monooxygenase.

The excellent mutants M3 and M10 of cyclohexanone monooxygenase obtained in example 1 were selected, and the excellent mutants M3 and M10 of cyclohexanone monooxygenase obtained in example 1 were added to 100mM Tris-HCl buffer (pH 9.0) containing 1mM, 2mM, 5mM of potentially chiral omeprazole sulfide, respectively, in an amount of 1g/L and 4g/L, and reacted at 30℃and pH 9.0 at 200rpm for 12 hours to obtain a reaction solution; in addition to the potential chiral omeprazole sulfide, the Tris-HCl buffer also contains coenzyme NADP with the concentration of 0.02mM ⁺ Glucose at a concentration of 1.5mM, glucose dehydrogenase GDH at a concentration of 1.5mM and methanol at a concentration of 5% (v/v).

The conversion rates of (S) -omeprazole and (R) -omeprazole produced by the cyclohexanone monooxygenase excellent mutant M3 and the cyclohexanone monooxygenase excellent mutant M10 asymmetric oxidation potential chiral omeprazole thioether at different reaction times are respectively detected, and the conversion efficiencies of (R) -omeprazole and (S) -omeprazole produced by the cyclohexanone monooxygenase excellent mutant M3 and the cyclohexanone monooxygenase excellent mutant M10 asymmetric oxidation potential chiral omeprazole thioether are calculated, and the detection results are shown in tables 5-6.

Tables 5 to 6 show that the excellent mutant M3 of cyclohexanone monooxygenase and the excellent mutant M10 of cyclohexanone monooxygenase reach a conversion rate of >99.9% at the time of reaction for 0.5h and 3h respectively, and the conversion efficiency of the excellent mutant M3 of cyclohexanone monooxygenase for producing (R) -omeprazole by asymmetric oxidation of latent chiral omeprazole thioether is obviously improved compared with that of the excellent mutant M10 of cyclohexanone monooxygenase; also, as can be seen from tables 3 to 4, when the added amount of the latent chiral omeprazole thioether is 5mM, the excellent mutant M10 of cyclohexanone monooxygenase cannot be completely converted, while the conversion rate of the excellent mutant M3 of cyclohexanone monooxygenase is not affected by the high concentration of the latent chiral thioether, and thus, the excellent mutant M3 of cyclohexanone monooxygenase can adapt to the environment of the high concentration of the latent chiral omeprazole thioether, asymmetrically oxidize the high concentration of the latent chiral omeprazole thioether to form (R) -omeprazole, and has a high conversion efficiency.

TABLE 5 conversion of Cyclohexanone monooxygenase excellent mutant M10 asymmetric oxidation potential chiral omeprazole thioether to (S) -omeprazole

TABLE 6 conversion of Cyclohexanone monooxygenase excellent mutant M3 asymmetric oxidation potential chiral omeprazole thioether to (R) -omeprazole

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of Jiangnan

<120> cyclohexanone monooxygenase mutant and application thereof

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<170> SIPOSequenceListing 1.0

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Gly Phe Gly Gly Ile Tyr Met Leu His Lys Leu Arg Asn Glu Leu Gly

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Leu Ser Val Thr Ala Phe Glu Lys Gly Gly Gly Val Gly Gly Thr Trp

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Tyr Phe Asn Arg Tyr Pro Gly Ala Lys Ser Asp Thr Glu Gly Phe Val

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Tyr Arg Tyr Ser Phe Asp Lys Asp Leu Leu Arg Glu Trp Asn Trp Thr

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Thr Arg Tyr Leu Glu Gln Ala Asp Val Leu Ala Tyr Leu Glu His Val

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Val Glu Arg Phe Asp Leu Gly Arg Asp Ile Arg Leu Asn Thr Glu Val

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Thr Gly Ala Val Phe Asp Glu Glu Ser Asp Leu Trp Thr Val Thr Thr

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Ala Thr Gly Glu Thr Thr Thr Ala Arg Tyr Leu Val Asn Ala Leu Gly

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Leu Leu Ala Lys Ser Asn Ile Pro Asp Ile Pro Gly Arg Asp Gly Phe

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Ala Gly Arg Leu Val His Thr Asn Ala Trp Pro Asp Asp Leu Asp Ile

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Thr Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Thr Gln

180 185 190

Phe Ile Ile Ala Ala Ala Lys Thr Ala Ser His Leu Thr Val Phe Gln

195 200 205

Arg Ser Pro Gln Tyr Cys Val Pro Ser Gly Asn Gly Pro Val Asp Gln

210 215 220

Thr Glu Val Asp Gly Thr Lys Glu Asn Phe Asp Ala Ile Trp Asp Gln

225 230 235 240

Val Arg Asn Ser Val Val Ala Phe Gly Phe Glu Glu Ser Gly Val Glu

245 250 255

Ala Met Ser Val Ser Glu Glu Glu Arg Arg Lys Val Phe Gln Glu Ala

260 265 270

Trp Asp Lys Gly Asn Gly Phe Arg Phe Met Phe Gly Thr Phe Cys Asp

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Ile Ala Thr Asn Pro Glu Ala Asn Ala Ala Ala Ala Ala Phe Ile Arg

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Ala Lys Ile Ala Glu Ile Val Asp Asp Pro Glu Thr Ala Arg Lys Leu

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Thr Pro Thr Asp Leu Tyr Ala Lys Arg Pro Leu Cys Asn Glu Gly Tyr

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Tyr Glu Thr Tyr Asn Arg Asp Asn Val Glu Leu Val Ser Ile Lys Glu

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Asn Pro Ile Arg Glu Ile Thr Pro Ala Gly Val Arg Thr Ala Asp Gly

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Thr Glu His Pro Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp Ala

370 375 380

Val Asp Gly Asn Tyr Arg Ala Met Asp Leu Arg Gly Arg Gly Gly Arg

385 390 395 400

His Ile Ser Glu His Trp Thr Gly Gly Pro Thr Ser Tyr Leu Gly Val

405 410 415

Ser Thr Ala Gly Phe Pro Asn Met Phe Met Ile Leu Gly Pro Asn Gly

420 425 430

Pro Phe Thr Asn Leu Pro Pro Ser Ile Glu Thr Gln Val Asp Trp Ile

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Ala Asp Met Thr Leu Phe Pro Lys Ala Asp Ser Trp Ile Phe Gly Ala

485 490 495

Asn Ile Pro Gly Lys Arg Asn Ala Val Met Phe Tyr Leu Ala Gly Leu

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Gly Phe Glu Leu Thr Arg Glu Asn Ala Thr Ala Ala Val

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atgagcacca cccatacccc ggatgtggat gccattgtta ttggtgcagg ttttggtggc 60

atttatatgc tgcataagct gcgcaatgag ctgggtctga gcgtgaccgc ttttgaaaaa 120

ggtggtggcg ttggcggtac atggtatttt aatcgttacc ctggtgcaaa gagcgatacc 180

gaaggttttg tttatcgcta tagcttcgac aaggacctgc tgcgcgaatg gaattggacc 240

acccgttatc tggaacaggc agatgtgctg gcatatctgg aacatgttgt ggaacgtttt 300

gacctgggcc gtgatattcg cctgaatacc gaagttaccg gcgccgtttt tgatgaagaa 360

agcgatctgt ggaccgttac caccgcaacc ggcgaaacca ccaccgcaag atatttagtg 420

aatgccctgg gtctgctggc aaaaagtaat attccggata ttccgggccg tgatggcttt 480

gcaggccgtt tagttcatac caatgcctgg ccggatgatc tggatattac cggcaaacgt 540

gttggcgtga ttggcaccgg cagcacaggt acacagttta ttattgccgc agccaaaacc 600

gcaagtcatc tgaccgtttt tcagcgtagc ccgcagtatt gtgttccgag cggcaatggt 660

ccggttgatc agaccgaagt tgatggcacc aaagaaaatt tcgacgcaat ttgggaccag 720

gtgcgtaata gtgttgtggc ctttggtttt gaagagagcg gcgttgaagc aatgagtgtg 780

agtgaagaag aacgccgtaa agtgtttcag gaagcatggg ataaaggcaa tggttttcgc 840

tttatgttcg gcaccttttg tgatattgcc accaatccgg aagccaatgc agcagccgcc 900

gcttttattc gtgcaaaaat tgcagaaatc gtggacgatc cggaaaccgc ccgcaaactg 960

acccctacag atttatatgc caagcgtccg ctgtgcaatg aaggttatta tgaaacctac 1020

aaccgtgaca acgtggaact ggtgagcatt aaagaaaacc cgattcgtga aatcaccccg 1080

gccggtgttc gtaccgcaga tggtacagaa catccgctgg atgttctggt gtttgccacc 1140

ggctttgatg ccgtggatgg taattatcgt gcaatggatc tgcgtggtcg tggtggtcgt 1200

catattagtg aacattggac cggcggcccg acctcatatc tgggtgttag caccgccggc 1260

tttccgaata tgtttatgat tctgggtcct aacggcccgt ttaccaatct gccgccgagt 1320

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cgtacagtgg aaccgacagc cgcagcagaa gaagcctgga cagcaacctg tgccgaaatt 1440

gcagatatga ccctgtttcc gaaagccgat agttggattt ttggtgcaaa cattccgggt 1500

aaacgcaatg cagtgatgtt ttatctggca ggcttaggtg catatcgtgc aaaactgcgt 1560

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gtgtaa 1626

Claims

1. A cyclohexanone monooxygenase mutant, characterized in that:

the mutant is characterized in that 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, and 435 th threonine is mutated into alanine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, and the 437 th leucine is mutated into cysteine;

or mutating 434 th phenylalanine of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine, mutating 435 th threonine into alanine, mutating 437 th leucine into cysteine, and mutating 507 th phenylalanine into valine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, and the 428 th leucine is mutated into glutamine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, and the 440 th serine is mutated into isoleucine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, the 440 th serine is mutated into isoleucine, and the 328 th lysine is mutated into cysteine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, the 440 th serine is mutated into isoleucine, the 328 th lysine is mutated into cysteine, and the 279 th phenylalanine is mutated into valine;

or the 434 th phenylalanine of the cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, the 440 th serine is mutated into isoleucine, the 328 th lysine is mutated into cysteine, the 279 th phenylalanine is mutated into valine, and the 290 th alanine is mutated into valine;

or the 434 th phenylalanine of cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, the 440 th serine is mutated into isoleucine, the 328 th lysine is mutated into cysteine, the 279 th phenylalanine is mutated into valine, the 290 th alanine is mutated into valine, the 245 th valine is mutated into alanine, and the 246 th valine is mutated into leucine;

or the 434 th phenylalanine of cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine, the 435 th threonine is mutated into alanine, the 437 th leucine is mutated into cysteine, the 507 th phenylalanine is mutated into valine, the 428 th leucine is mutated into glutamine, the 440 th serine is mutated into isoleucine, the 328 th lysine is mutated into cysteine, the 279 th phenylalanine is mutated into valine, the 290 th alanine is mutated into valine, the 245 th valine is mutated into alanine, the 246 th valine is mutated into leucine, the 491 th serine is mutated into cysteine, and the 492 th tryptophan is mutated into arginine.

2. A gene encoding the cyclohexanone monooxygenase mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A host cell expressing the cyclohexanone monooxygenase mutant of claim 1.

5. The host cell of claim 4, wherein: the host cell is a bacterial, fungal, plant cell or animal cell.

6. Use of a cyclohexanone monooxygenase mutant according to claim 1, a gene according to claim 2, a recombinant plasmid according to claim 3 or a host cell according to claim 4 or 5 for the production of chiral sulfoxides.

7. The use according to claim 6, characterized in that: the application is that the cyclohexanone monooxygenase mutant is added into a reaction system containing the potential chiral thioether for reaction to obtain the chiral sulfoxide.

8. The use according to claim 7, characterized in that: the latent chiral thioether comprises phenyl sulfide, 2-methylthionaphthalene, benzyl phenyl sulfide or omeprazole sulfide.

9. The use according to claim 7, characterized in that: the chiral sulfoxide comprises benzyl sulfoxide, 2- (methylsulfinyl) naphthalene, benzyl phenyl sulfoxide or chiral omeprazole.

10. The use according to claim 7, characterized in that: the reaction temperature is 30-35 ℃, and the pH is 7-9.