CN115044566A

CN115044566A - Cyclohexanone monooxygenase mutant and application thereof

Info

Publication number: CN115044566A
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: 2022-09-13
Anticipated expiration: 2042-06-29
Also published as: CN115044566B

Abstract

The invention discloses a cyclohexanone monooxygenase mutant and application thereof, belonging to the technical field of enzyme engineering and microbial engineering. The cyclohexanone monooxygenase mutant disclosed by the invention has higher activity on various latent chiral thioethers, especially for omeprazole thioethers, under the condition that wild cyclohexanone monooxygenase cannot convert the substrate to produce chiral omeprazole, the mutant disclosed by the invention can asymmetrically oxidize the latent chiral omeprazole thioethers to produce chiral omeprazole, the catalytic efficiency is higher, the ee value can reach 99%, and meanwhile, the mutant also has higher conversion efficiency on other latent chiral thioethers, and has a 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 field of enzyme engineering and microbial engineering.

Background

The 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, esomeprazole), chemically (S) -5-methoxy-2- [ [ (4-methoxy-3, 5-dimethyl-2-pyridyl) 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 the first optically pure proton pump inhibitor on the market. Compared with racemate omeprazole, the esomeprazole has better acid inhibiting effect. 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 method for producing the chiral drug esomeprazole by a biological catalysis method has huge market space in the pharmaceutical industry. At present, the chiral drug esomeprazole is mainly produced at home and abroad by a chemical method, and the esomeprazole produced by a biological method is still in a laboratory research stage. More researches on the production of esomeprazole by a bio-enzyme catalysis method abroad are carried out and certain research results are obtained, and a large gap still exists in domestic researches on the aspect.

In 2013, Codexis used for engineering cyclohexanone monooxygenase mutants with more than 30 mutation sites, 100g/L of omeprazole thioether substrate is added into a 75mL reaction system, the mixture is degassed and aerated for 4 times, and the mixture is subjected to batch feeding of enzyme and coenzyme and stirring reaction in an oxygen atmosphere, and co-transformation is carried out for 69 hours at the rotation speed of 200rpm, so that the transformation rate is 99.6%, and the e.e. value is 99.9%. In 2011, Peter, Eva and the like isolated a lysine bacillus oxidizing omeprazole thioether. By adopting a mode of feeding enzyme and substrate in batches, the added bacteria amount is 25mg CDW in each 5mL LB culture medium, the total adding amount of the substrate is 0.1g/L, the flask is shaken at 200rpm for 24h for conversion, and finally the conversion rate of 77% is obtained, and the e.e. value is 100%. In 2019, an engineered cyclohexanone monooxygenase mutant with more than 15 mutation sites is constructed by allowable construction and team, and omeprazole thioether is selectively oxidized to generate the required (S) -omeprazole. Omeprazole thioether loading was 15mM substrate, complete conversion within 6 h. The CHMO has huge application potential in the field of chiral drug synthesis, including esomeprazole in the research, and the patents of optically pure esomeprazole salts (including sodium salt, magnesium salt, lithium salt, potassium salt, calcium salt and the like) which are critical in 5 months in 2014 are due, so that the CHMO faces the market with rich profits, but the CHMO has the defects of complex process, environmental pollution, more byproducts, low optical selectivity and the like, and the esomeprazole produced by a biological method is more and more concerned due to the advantages of environment friendliness, mild conditions, strong characteristics and the like. Cyclohexanone monooxygenase has the potential for industrial production due to its high stereoselectivity for omeprazole thioether.

Therefore, more cyclohexanone monooxygenases for producing chiral esomeprazole by asymmetrically oxidizing latent chiral omeprazole thioether are urgently needed to realize large-scale industrial production of chiral esomeprazole as well as large-scale application of chiral esomeprazole in the field of medicine.

Disclosure of Invention

In order to solve the problems, the invention provides 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 invention aims to provide a cyclohexanone monooxygenase mutant, wherein the mutant is obtained by mutating phenylalanine 434 of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine (F434I), and simultaneously mutating threonine 435 into alanine (T435A), so that the mutant F434I/T435A is obtained.

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

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

Furthermore, the mutant is obtained by mutating phenylalanine 434 of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine (F434I), threonine 435 into alanine (T435A), leucine 437 into cysteine (L437C), phenylalanine 507 into valine (F507V), and leucine 428 into glutamine (L428Q), so that mutant F434I/T435A/L437C/F507V/L428Q is obtained.

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

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

Furthermore, the mutant is obtained by mutating phenylalanine 434 of cyclohexanone monooxygenase with an amino acid sequence shown as SEQ ID NO.1 into isoleucine (F434I), threonine 435 into alanine (T435A), leucine 437 into cysteine (L437C), phenylalanine 507 into valine (F507V), leucine 428 into glutamine (L428Q), serine 440 into isoleucine (S440I), lysine 328 into cysteine (K328C), and phenylalanine 279 into valine (F279V), so that mutant F434I/T435A/L437C/F507V/L Q/S82428/K328C/F279V is obtained.

Furthermore, the mutant is obtained by mutating phenylalanine 434 to isoleucine (F434I), threonine 435 to alanine (T435A), leucine 437 to cysteine (L437C), phenylalanine 507 to valine (F507V), leucine 428 to glutamine (L428Q), serine 440 to isoleucine (S440I), lysine 328 to cysteine (K328C), phenylalanine 279 to valine (F279V), and alanine 290 to valine (A290V), which have amino acid sequences shown in SEQ ID NO.1, thereby obtaining mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V/A290V.

Furthermore, the mutant is obtained by mutating phenylalanine 434 of cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 into isoleucine (F434I), threonine 435 into alanine (T435A), leucine 437 into cysteine (L437C), phenylalanine 507 into valine (F507V), leucine 428 into glutamine (L428Q), serine 440 into isoleucine (S440I), lysine 328 into cysteine (K328C), phenylalanine 279 into valine (F279V), alanine 290 into valine (A290V), valine 245 into alanine (V245A), meanwhile, valine at position 246 is mutated into leucine (V246L), and a mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V/A290V/V245A/V246L is obtained.

Furthermore, the mutant is obtained by mutating phenylalanine 434 to isoleucine (F434I), threonine 435 to alanine (T435A), leucine 437 to cysteine (L437C), phenylalanine 507 to valine (F507V), leucine 428 to glutamine (L428Q), serine 440 to isoleucine (S440I), lysine 328 to cysteine (K328C), phenylalanine 279 to valine (F279V), alanine 290 to valine (A290V), valine 245 to alanine (V245A), valine 246 to leucine (V865246) 246 to serine 491 to cysteine (S36491), tryptophan 36492) to arginine 36492, the mutant F434I/T435A/L437C/F507V/L428Q/S440I/K328C/F279V/A290V/V245A/V246L/S491C/W492R is obtained.

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

The third purpose of the invention is to provide a recombinant plasmid carrying the gene.

Further, the vector of the recombinant plasmid 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 mutant.

Further, the host cell is a bacterium, a fungus, a plant cell or an animal cell.

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

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

The fifth purpose of the invention is to provide the application of the cyclohexanone monooxygenase mutant, the gene, the expression vector or the host cell in the production of chiral sulfoxide.

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

Further, the latent chiral thioether is thioanisole, 2-methylthionaphthalene, benzylphenylsulfide or omeprazole thioether.

Further, the chiral sulfoxide is benzyl sulfoxide, 2- (methylsulfinyl) naphthalene, benzyl phenyl sulfoxide or chiral omeprazole. Specifically, when the chiral methyl sulfide is latent, the chiral sulfoxide is methyl sulfoxide; when the chiral 2-methylthio naphthalene is latent, the chiral sulfoxide is 2- (methylsulfinyl) naphthalene; when the benzyl phenyl sulfide is latent chirality, the chirality sulfoxide is benzyl phenyl sulfoxide; when the chiral omeprazole thioether is latent chiral omeprazole, 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 circulating system; the coenzyme cycle system comprises D-glucose and glucose dehydrogenase, or formate and formate dehydrogenase, or lactate and lactate dehydrogenase, or glycerol and glycerol dehydrogenase.

Further, the coenzyme is NADP ⁺ 、NADPH、NAD ⁺ And one or more of NADH.

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

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

Furthermore, in the reaction system, the concentration of the coenzyme is 0.1-1 mmol/L.

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

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

Further, the reaction system is a buffer solution containing the latent chiral thioether, the coenzyme and a coenzyme circulating system.

Further, the buffer is Tris-HCl buffer.

Furthermore, 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 value is 7-9.

The invention has the beneficial effects that:

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

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

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

(4) The cyclohexanone monooxygenase mutant has high catalytic efficiency in producing chiral sulfoxide by asymmetrically oxidizing latent chiral thioether, wherein the catalytic efficiency of the mutant M10 in producing chiral (S) -omeprazole by asymmetrically oxidizing latent chiral omeprazole thioether is 0.38min ^–1 ·mM ^–1 The ee value 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 latent chiral 2-methylthionaphthalene and benzyl phenyl thioether, and the ee values can respectively reach 99 percent (R) and 99 percent (S); the cyclohexanone monooxygenase mutant of the invention can also asymmetrically oxidize latent chiral 2-methylthionaphthalene and benzyl phenyl sulfide, wherein the mutant M2-M6 can asymmetrically oxidize the latent chiral 2-methylthionaphthalene and benzyl phenyl sulfide, and the ee values can respectively reach 99% (R) and 99% (S). The activity and the selectivity can be kept consistent with those of the wild type.

(6) The cyclohexanone monooxygenase mutant has high catalytic efficiency in the production of chiral sulfoxide by asymmetrically oxidizing latent chiral thioether, so the cyclohexanone monooxygenase mutant has extremely high application prospect in the production of chiral sulfoxide such as (S) -omeprazole, (R) -omeprazole and the like.

Drawings

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

FIG. 2 shows the SDS-PAGE electrophoretic analysis of the expression product obtained by shake flask induction fermentation of recombinant E.coli; wherein, M: standard protein marker, 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 pure enzyme of wild type WT and mutant M1-M10 obtained by shake flask induction fermentation.

FIG. 3 is a chiral chromatogram of the products (R) -omeprazole and (S) -omeprazole obtained by asymmetrically oxidizing latent chiral omeprazole thioether with mutants M3 and M10.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Coli BL21(DE3) referred to in the examples below was purchased from north na; the pET-28a (+) plasmid, NADPH, referred to in the examples below were purchased from Novagen; glucose Dehydrogenase (GDH) and Lactate Dehydrogenase (LDH) mentioned in the following examples were obtained from nunoprazan; d-glucose referred to in the examples below was purchased from Czech; the latent chiral thioethers referred to in the following examples are purchased from the national pharmaceutical group (the above-mentioned strain escherichia coli e. coli BL21(DE3) is commercially available and does not require a deposit for the patent procedure).

The media involved in the following examples are as follows:

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 agar powder and 50 mg/L kanamycin ^-1 。

The detection methods referred to in the following examples are as follows:

the method for detecting the enzyme activity of the cyclohexanone monooxygenase comprises the following steps:

carrying out heat preservation on Tris-HCl buffer solution (100mM, pH 9.0) containing 1mM NADPH and 1.0mM substrate latent chiral omeprazole thioether at 30 ℃ for 2min, adding 10 mu L of pure enzyme solution into the Tris-HCl buffer solution to carry out reaction at 30 ℃ for 30min, stopping the reaction by using acetonitrile with the same volume, measuring the generation amount of a product by using high performance liquid chromatography, and calculating the enzyme activity according to the generation amount;

the calculation formula of the enzyme activity is as follows:

the enzyme activity (u/mg) ═ C1 XV 1 X10 ³ /(T×C2×V2)；

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

definition of enzyme activity: under the condition, the enzyme amount required for catalytically oxidizing one mu mol of substrate per minute to produce one mu mol of product is one enzyme activity unit (1 u).

The method for detecting the conversion efficiency and stereoselectivity of the cyclohexanone monooxygenase asymmetric oxidation of latent chiral omeprazole thioether to generate chiral (S) -omeprazole and (R) -omeprazole is as follows:

performing heat preservation on Tris-HCl buffer solution (100mM, pH 9.0) containing 1mM NADPH and 1.0mM substrate latent chiral omeprazole thioether at 30 ℃ for 2min, and adding 10 mu L of pure enzyme solution into the Tris-HCl buffer solution to react at 30 ℃ for 60 min; after the reaction is finished, adding 500 mu L of ethyl acetate, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifugal tube, adding 500 mu L of chromatographic pure ethanol when the organic phase is completely volatilized naturally, and carrying out chiral liquid chromatography to analyze the conversion efficiency and the ee value;

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

the stereoselective chromatographic conditions were as follows: daicel Chiralcel AD-3(5 μm, 250 mm. times.4.6 mm) liquid chromatography column, mobile phase is n-hexane: isopropanol: acetic acid (50:50:0.01, v/v/v), flow rate is 0.5mL/min, column temperature is 30 ℃, ultraviolet detection wavelength is 250nm, sample injection amount is 10 μ L, (S) -omeprazole and (R) -omeprazole retention time are 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. the _R : in the reaction solution(R) -molar concentration of omeprazole; a. the _sub : the molar concentration of the unreacted omeprazole thioether in the reaction solution.

Example 1 construction, expression and purification of Cyclohexanone monooxygenase mutants

Chemically synthesizing a gene (the nucleotide sequence of the gene is shown as SEQ ID NO. 2) of cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO. 1; connecting the obtained gene with pET-28a (+) plasmid after double enzyme digestion (Nde I and Xho I), transforming Escherichia coli E.coli BL21(DE3) by using a connection product, coating the transformation product on an LB solid culture medium, culturing for 8-10 h at 37 ℃, selecting 5 transformants on the LB solid culture medium, inoculating the transformants into an LB liquid culture medium for culturing, extracting the plasmids after culturing for 10h at 37 ℃, performing enzyme digestion verification and sequencing verification on the extracted plasmids, and obtaining the recombinant plasmid pET28a-AmCHMO containing the gene coding the wild cyclohexanone monooxygenase and the recombinant bacterium E.coli BL21/pET28a-AmCHMO containing the gene coding the wild cyclohexanone monooxygenase after verification is correct.

The whole plasmid PCR technology is utilized, the obtained recombinant plasmid pET28a-AmCHMO is taken as a template for site-directed mutagenesis, obtaining a mutant containing encoded cyclohexanone monooxygenase F434I (phenylalanine at position 434 is mutated into isoleucine), T435A (threonine at position 435 is mutated into alanine), L437C (leucine at position 437 is mutated into cysteine), F507V (phenylalanine at position 507 is mutated into valine), L428Q (leucine at position 428 is mutated into glutamine), S440I (serine at position 440 is mutated into isoleucine), K328C (lysine at position 328 is mutated into cysteine), F279V (phenylalanine at position 279 is mutated into valine), A290V (alanine at position 290 is mutated into valine), V245A/V246L (valine at position 245, two valine at position 246 are mutated into alanine and leucine respectively), S491C/W492R (serine 491 at position 492 and tryptophan are mutated into cysteine and arginine);

mutant site and numbering: F434I/T435A (M1), F434I/T435A/L437C (M2), F434I/T435A/L437C/F507V (M3), F434I/T435A/L437C/F507V/L428Q (M4), F434I/T435A/L437C/F507V/L V/S440V (M V), F434V/T435V/L V/F507V/L V/V (M V), F434/T435/L V/L437/V/F507/K V/F507/L V/F V/L V/F V/F V/3678/V/L3678 (M36437/L V/3/V/3/L V/L V/L V/L V/L V/L V/L V/L/V/L V/L V/L/V/F/V/L V/L V/L/V/L/V/L V/L/V/L/V/3678 (M/L/V/L/V/L/V/L/36, the cyclohexanone monooxygenase mutants are named as M1-M10 respectively;

the primers used for the mutations F434I, T435A, L437C, F507V, L428Q, S440I, K328C, F279V, A290V, V245A/V246L and 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 μ L of KOD enzyme (2.5U/mL), l.0 μ L of template (5-50 ng), 4.0 μ L of dNTP, 5.0 μ L of 10 × reaction buffer, 1.0 μ L of each of upstream and downstream primers, and up to 50 μ L of ddH 2O;

the PCR product amplification conditions were all: (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 the steps (2) - (4) for 10-15 cycles, finally extension at 72 ℃ for 10min, and storing the PCR amplification product at 4 ℃.

Detecting PCR amplification products by using 1% agarose gel electrophoresis, after the detection is finished, adding 0.5 mu L of methylated template digestive enzyme (Dpn I) into 10 mu L of amplification products, blowing and sucking a gun head for uniformly mixing, reacting for 1h at 37 ℃, converting the amplification products treated by the Dpn I into escherichia coli E.coli BL21(DE3), coating the conversion products on an LB solid culture medium, culturing for 8-10 h at 37 ℃, selecting 3 transformants on the LB solid culture medium, inoculating the transformants into an LB liquid culture medium for culturing, extracting plasmids after culturing for 10h at 37 ℃, carrying out enzyme digestion verification (the verification result can be shown in figure 1) and sequencing verification, and obtaining the recombinant plasmid T28-AmCHMO-1-recombinant plasmid T a-AmCHMO-10 respectively containing genes coding cyclohexanone monooxygenase mutants M1-M10 and the recombinant plasmid E. 21/coli/E.3526 coli respectively containing genes coding cyclohexanone monooxygenase M1-M10 by verifying that the plasmids are correct AmCHMO-1-recombinant strain E.coli BL21/pET28 a-AmCHMO-10.

Respectively coating the obtained recombinant bacterium E.coli BL21/pET28 a-AmCHMO-1-recombinant bacterium E.coli BL21/pET28a-AmCHMO-10 on an LB solid culture medium, and culturing at 37 ℃ for 8-10 h to obtain a single colony; selecting a single colony, inoculating the single colony into an LB liquid culture medium, and culturing at 37 ℃ for 12-14 h to obtain a seed solution; inoculating the seed solution into LB liquid culture medium according to the inoculum size of 2% (v/v), culturing at 37 deg.C and 200rpm to OD ₆₀₀ After reaching 0.8, adding IPTG with the final concentration of 0.2mM into the fermentation liquor, and continuing to perform induction culture for 8 hours at 25 ℃ to obtain the fermentation liquor; centrifuging the fermentation liquid at 4 deg.C and 8000rpm for 10min, and collecting cells; the collected cells were suspended in potassium phosphate buffer (100 mmol. multidot.L) ^-1 pH 6.0), collecting fractions containing wild-type cyclohexaneAnd (3) cell disruption supernatant of ketone monooxygenase and cyclohexanone monooxygenase mutants M1-M10.

The cell disruption supernatant obtained was purified using affinity column HisTrap FF crude (nickel column) as follows: buffer A (20 mmol. L) was used first ^-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, continuing to elute proteins not bound to the nickel column using buffer a, after the penetrating peak has drained, from buffer a to buffer B (20mmol · L) ^-1 Tris-HCl，500mmol·L ^- ¹ NaCl，500mmol·L ^-1 Imidazole, pH 7.4), eluting the recombinant protein bound on the nickel column to obtain pure enzyme solution of wild cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M1-M10.

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

As can be seen from FIG. 2, the pure enzyme solutions of the cyclohexanone monooxygenase mutants M1-M10 all showed a single band at about 59kDa, and had fewer hetero-proteins, indicating that the nickel column purification effect is better.

Example 2 kinetic parameters of Cyclohexanone monooxygenase mutants and stereoselectivity of asymmetric Oxidation of latent chiral thioether to chiral sulfoxide

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

As can be seen from Table 1, the catalytic efficiency of (R) -omeprazole generated by asymmetrically oxidizing latent chiral omeprazole thioether by using cyclohexanone monooxygenase mutant M3 is remarkably improved compared with that of wild cyclohexanone monooxygenase; compared with the cyclohexanone monooxygenase mutant M7, the catalytic efficiency of the cyclohexanone monooxygenase mutant M8 for asymmetrically oxidizing latent chiral omeprazole thioether to generate (S) -omeprazole is improved by 10 times; compared with the cyclohexanone monooxygenase mutant M8, the catalytic efficiency of the cyclohexanone monooxygenase mutant M10 for asymmetrically oxidizing latent chiral omeprazole thioether to generate (S) -omeprazole is improved by 5 times.

The stereoselectivity and relative activity of the wild-type cyclohexanone monooxygenase obtained in example 1 and the cyclohexanone monooxygenase mutants M2-M10 for asymmetrically oxidizing latent chiral thioether to generate chiral sulfoxide are detected, and the detection results are shown in Table 2.

As can be seen from Table 2, the stereoselectivity of the cyclohexanone monooxygenase mutants M2-M6 for asymmetrically oxidizing latent chiral thioanisole to generate chiral sulfoxide is not reduced compared with that of the wild cyclohexanone monooxygenase; but the relative activity is gradually reduced, and the activity of asymmetrically oxidizing the latent chiral thioanisole is lost when the cyclohexanone monooxygenase mutants M7-M10 are reached; as can be seen from Table 2, the wild cyclohexanone monooxygenase can asymmetrically oxidize latent chiral 2-methylthionaphthalene, and the stereoselectivity of the cyclohexanone monooxygenase mutant M2-M5 for asymmetrically oxidizing latent chiral thioanisole to generate chiral sulfoxide is not reduced compared with the wild cyclohexanone monooxygenase, the ee value can reach 99% (R), and the activity is basically kept unchanged; compared with wild cyclohexanone monooxygenase, when the cyclohexanone monooxygenase mutant M6-M10 asymmetrically oxidizes latent chiral 2 methylthio naphthalene, the selectivity is reduced, and the activity is firstly reduced and then increased; for benzylphenylsulfide, the selectivity of cyclohexanone monooxygenase mutants M3 to M6, except for cyclohexanone monooxygenase mutant M2, was not decreased compared to wild-type cyclohexanone monooxygenase; for omeprazole thioether, the activity of chiral omeprazole generated by asymmetrically oxidizing latent chiral omeprazole ether by cyclohexanone monooxygenase mutants M2-M10 is obviously improved compared with that of wild cyclohexanone monooxygenase; wherein, the activity of the cyclohexanone monooxygenase mutant M2-M3 for asymmetrically oxidizing latent chiral omeprazole thioether to generate chiral (R) -omeprazole is gradually improved, and the selectivity is kept unchanged. The stereoselectivity of the chiral omeprazole generated by asymmetrically oxidizing latent chiral omeprazole ether by cyclohexanone monooxygenase mutants M4-M6 is gradually reduced; and the relative activity is gradually reduced, and the activity is reduced to the minimum when reaching 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 mutant 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 activity was detected.

TABLE 2 relative activity and stereoselectivity of the asymmetric oxidation of latent chiral thioethers to chiral sulfoxides by wild-type cyclohexanone monooxygenase and cyclohexanone monooxygenase mutants M2-M10

ND, no activity is detected.

Example 3 conversion efficiency of Cyclohexanone monooxygenase mutants to sulfoxide by asymmetric Oxidation of latent chiral thioether

Selecting the cyclohexanone monooxygenase mutant M1-M10 obtained in example 1, adding the cyclohexanone monooxygenase mutant M1-M10 obtained in example 1 to 100mM Tris-HCl buffer (pH 9.0) respectively containing 5mM, latent chiral thioanisole, 2-methylthionaphthalene and benzyl phenyl sulfide at an addition amount of 2g/L, and reacting at 30 ℃, pH 9.0 and 200rpm for 1-12h to obtain a reaction solution; in addition to the latent chiral omeprazole thioether, the Tris-HCl buffer also contains coenzyme NADP + at a 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).

And respectively detecting the conversion rate and selectivity of the cyclohexanone monooxygenase mutant M1-M10 for asymmetrically oxidizing the latent chiral methyl sulfide, 2-methylthionaphthalene and benzyl phenyl sulfide into corresponding sulfoxide at different times, wherein the detection results are shown in Table 3.

TABLE 3 conversion of Cyclohexanone monooxygenase mutants M1-M10 to the asymmetric oxidation of latent chiral thioanisole, 2-methylthionaphthalene, benzylthioanisole to the corresponding sulfoxides

ND, no activity is detected.

As can be seen from Table 3, at 5mM substrate concentration, cyclohexanone monooxygenase mutants M1-M10 reach > 90% conversion rate in reaction for 1-12h, respectively, wherein the conversion rate of the cyclohexanone monooxygenase mutants M1-M3 for asymmetrically oxidizing latent chiral methyl phenyl sulfide, 2-methylthionaphthalene and benzyl phenyl sulfide into corresponding sulfoxide is consistent with that of wild-type cyclohexanone monooxygenase. And compared with the conversion efficiency of wild cyclohexanone monooxygenase, the conversion rate of the cyclohexanone monooxygenase mutant M4-M10 for asymmetrically oxidizing latent chiral methyl phenyl sulfide, 2-methylthionaphthalene and benzyl phenyl sulfide into corresponding sulfoxide is obviously reduced. The above results indicate that the substrate binding pocket of the mutant is gradually enlarged and loses catalytic activity to a substrate with smaller volume as the mutation site is increased compared with the wild-type cyclohexanone monooxygenase.

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

Selecting the cyclohexanone monooxygenase mutant M1-M10 obtained in example 1, adding the cyclohexanone monooxygenase mutant M1-M10 obtained in example 1 into 100mM Tris-HCl buffer solutions (pH 9.0) respectively containing 2mM and latent chiral omeprazole thioether at the addition amount of 2g/L, and reacting for 1-12h at 30 ℃, pH 9.0 and 200rpm to obtain reaction liquid; in addition to the latent chiral omeprazole thioether, the Tris-HCl buffer also contains coenzyme NADP + at a 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).

Respectively detecting the conversion rate and selectivity of the cyclohexanone monooxygenase mutant M1-M10 for asymmetrically oxidizing the latent chiral omeprazole thioether into (S) -omeprazole and (R) -omeprazole when the reaction is carried out for different time, calculating the conversion efficiency of the cyclohexanone monooxygenase mutant M3 and the cyclohexanone monooxygenase mutant M10 for asymmetrically oxidizing the latent chiral omeprazole thioether into (R) -omeprazole and (S) -omeprazole, and the detection results are shown in Table 4.

TABLE 4 conversion of Cyclohexanone monooxygenase mutant M1-M10 to (R) -omeprazole or (S) -omeprazole by asymmetric oxidation of latent chiral omeprazole thioether

ND, no activity is detected.

As can be seen from table 4, at a substrate concentration of 2mM, the cyclohexanone monooxygenase mutants M1-M10 reach a conversion rate of > 99% in the reaction time of 1-12h, respectively, while the wild-type cyclohexanone monooxygenase is not converted, and thus, the conversion efficiency of the cyclohexanone monooxygenase mutants M1-M10 for asymmetrically oxidizing latent chiral omeprazole sulfide into (R) -omeprazole and (S) -omeprazole is remarkably improved compared with the conversion efficiency of the wild-type cyclohexanone monooxygenase for asymmetrically oxidizing latent chiral omeprazole sulfide.

The excellent cyclohexanone monooxygenase mutants M3 and M10 obtained in example 1 were selected, the excellent cyclohexanone monooxygenase mutant M3 and the excellent cyclohexanone monooxygenase mutant M10 obtained in example 1 were added to 100mM Tris-HCl buffer solutions (pH 9.0) containing 1mM, 2mM and 5mM latent chiral omeprazole thioether respectively at the addition amounts of 1g/L and 4g/L, and reacted at 30 ℃, pH 9.0 and 200rpm for 12 hours to obtain reaction solutions; besides the latent chiral omeprazole thioether, the Tris-HCl buffer solution 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).

Respectively detecting the conversion rate of the excellent cyclohexanone monooxygenase mutant M3 and the excellent cyclohexanone monooxygenase mutant M10 in the process of asymmetrically oxidizing latent chiral omeprazole thioether to generate (S) -omeprazole and (R) -omeprazole in different reaction times, and calculating the conversion efficiency of the excellent cyclohexanone monooxygenase mutant M3 and the excellent cyclohexanone monooxygenase mutant M10 in the process of asymmetrically oxidizing latent chiral omeprazole thioether to generate (R) -omeprazole and (S) -omeprazole, wherein the detection results are shown in tables 5-6.

From tables 5 to 6, under the concentration of 1mM substrate, the excellent cyclohexanone monooxygenase mutant M3 and the excellent cyclohexanone monooxygenase mutant M10 reach the conversion rate of more than 99.9% in the reaction of 0.5h and 3h, respectively, so that the conversion efficiency of the excellent cyclohexanone monooxygenase mutant M3 for asymmetrically oxidizing latent chiral omeprazole sulfide to generate (R) -omeprazole is obviously improved compared with the conversion efficiency of the excellent cyclohexanone monooxygenase mutant M10 for asymmetrically oxidizing latent chiral omeprazole sulfide to generate (S) -omeprazole; in addition, as can be seen from tables 3 to 4, when the addition amount of the latent chiral omeprazole sulfide is 5mM, the cyclohexanone monooxygenase excellent mutant M10 cannot be completely converted, and the conversion rate of the cyclohexanone monooxygenase excellent mutant M3 is not affected by high-concentration latent chiral sulfide, so that the cyclohexanone monooxygenase excellent mutant M3 can adapt to the environment of high-concentration latent chiral omeprazole sulfide, asymmetrically oxidize the high-concentration latent chiral omeprazole sulfide to generate (R) -omeprazole, and has high conversion efficiency.

TABLE 5 conversion of Cyclohexanone monooxygenase Excellent mutant M10 asymmetric Oxidation of latent chiral omeprazole thioether to (S) -omeprazole

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

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of south of the Yangtze river

<120> cyclohexanone monooxygenase mutant and application thereof

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Met Ser Thr Thr His Thr Pro Asp Val Asp Ala Ile Val Ile Gly Ala

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

275 280 285

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

290 295 300

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

305 310 315 320

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

340 345 350

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

355 360 365

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

435 440 445

Gly Glu Leu Ile Arg His Ala Glu Arg Thr Gly Val Arg Thr Val Glu

450 455 460

Pro Thr Ala Ala Ala Glu Glu Ala Trp Thr Ala Thr Cys Ala Glu Ile

465 470 475 480

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 Ala Tyr Arg Ala Lys Leu Arg Glu Val Ala Asp Ala Gly Tyr Thr

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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

attgaaaccc aggttgattg gattggcgaa ctgattcgcc atgcagaacg caccggcgtt 1380

cgtacagtgg aaccgacagc cgcagcagaa gaagcctgga cagcaacctg tgccgaaatt 1440

gcagatatga ccctgtttcc gaaagccgat agttggattt ttggtgcaaa cattccgggt 1500

aaacgcaatg cagtgatgtt ttatctggca ggcttaggtg catatcgtgc aaaactgcgt 1560

gaagtggcag atgccggtta taccggcttt gaactgaccc gtgaaaatgc aaccgccgca 1620

gtgtaa 1626

Claims

1. A cyclohexanone monooxygenase mutant, characterized in that:

the mutant is obtained by mutating phenylalanine 434 of cyclohexanone monooxygenase with the amino acid sequence shown as SEQ ID NO.1 into isoleucine, and simultaneously mutating threonine 435 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 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, and the 507 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, 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 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, 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 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, 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 bacterium, a fungus, a plant cell or an animal cell.

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

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

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

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

10. Use according to claim 6, characterized in that: the reaction temperature is 30-35 ℃, and the pH is 7-9.