Sulfide monooxygenase mutants and their use in the preparation of chiral loprazol drugs

Abstract

The application belongs to the technical field of bioengineering, and relates to a thioether monooxygenase mutant, a nucleic acid for coding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, and application of the recombinant thioether monooxygenase mutant in preparation of a chiral lansoprazole medicine. Compared with other biological catalysts for preparing optically pure lansoprazole medicines, the thioether monooxygenase mutant provided by the application has the advantages of high protein expression amount, high catalytic activity, strong solvent tolerance, good thermal stability, high optical purity of a product, and high yield, and shows a wide application prospect in industrial application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a thioether monooxygenase mutant, a nucleic acid encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, the preparation of the mutant enzyme preparation, and the application of the mutant enzyme preparation in the preparation of chiral proton pump inhibitors. Background Technology

[0002] (S)-Omeprazole is a proton pump inhibitor (PPI) for treating acid-producing digestive disorders. Approved for marketing in 2001, it is the S-isomer of omeprazole and was the first chiral PPI to appear on the market. Developed by AstraZeneca, its chemical name is S-5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole. Currently, the form sold in hospitals and pharmacies is mostly (S)-omeprazole magnesium trihydrate, whose properties and structural characteristics have been thoroughly studied. Clinical practice has shown that (S)-omeprazole is safer and more effective than omeprazole, with better clinical efficacy, better control of gastric acid secretion, and relief of gastric symptoms. Furthermore, (S)-omeprazole has a longer duration of action and does not require frequent dosing, making it more popular with patients and doctors. Due to its wide application and good clinical efficacy, (S)-omeprazole has become one of the world's best-selling drugs, making a significant contribution to the treatment of digestive system diseases and occupying a pivotal position in the pharmaceutical market.

[0003] Currently, (S)-omeprazole is industrially synthesized entirely by chemical methods. However, the problems commonly encountered in chemical synthesis, such as expensive chiral catalysts and poor stereoselectivity, cannot be avoided. Furthermore, the large amounts of peroxyacids, H₂O₂, organic catalysts, and organic solvents used in chemical synthesis can cause serious harm to the health of workers and the environment. Chemical reaction conditions are relatively harsh, requiring sophisticated production equipment and increasing production costs. In response to the call for green living and green manufacturing, biosynthesis, with its advantages of good catalytic selectivity, mild reaction conditions, and environmentally friendly reaction systems, is gradually becoming a beneficial supplement to chemical synthesis.

[0004] Currently, the main biosynthetic route for (S)-omeprazole uses prechiral thioethers as substrates and whole cells and free enzymes as catalysts to catalyze their asymmetric oxidation, yielding optically pure (S)-omeprazole. Currently reported biocatalysts for the synthesis of (S)-omeprazole mainly include: 1) the fungus *Cunninghamella echinulata* MK40, which can catalyze the asymmetric oxidation of 7.5 mM omeprazole sulfide, but the conversion rate is only 45%; 2) *Lysinibacillus* sp. B71 cells, which can catalyze the production of (S)-omeprazole from 0.1 g / L omeprazole sulfide, but the conversion rate is also only 70%; 3) PAMO derived from *Limnobacter* sp. can generate (S)-omeprazole through whole-cell asymmetric oxidation of 50 mM omeprazole sulfide; 4) in the patent WO2011071982 published by Codexis in the United States, the authors modified *Acinetobacter calcoaceticus* NCIMB through directed evolution. The cyclohexanone monooxygenase mutant of 9871 can catalyze the synthesis of (S)-omeprazole from 100 g / L omeprazole thioether, but it requires adding lyophilized enzyme powder in 5 batches; 5) Patent CN108118035A discloses a mutant that can catalyze the synthesis of (S)-omeprazole from 165 g / L omeprazole thioether, but these patents have problems such as large amount of extraction enzyme used, poor tolerance, complicated catalyst preparation, and difficult separation and extraction; 6) Patent CN113583985A and related literature report the realization of yeast extracellular secretion expression, which can catalyze 10 g / L omeprazole thioether. Its catalyst preparation is simple, but there are still problems such as low enzyme expression level, low oxidation activity, insufficient stability, and low substrate loading, which are difficult to meet the requirements of industrial production. Summary of the Invention

[0005] To address the problems of low enzyme expression, low oxidative activity, insufficient stability, and low substrate loading in the synthesis of the chiral sulfoxide drug (S)-omeprazole using yeast secretory expression of thioether monooxygenase, this invention provides a thioether monooxygenase mutant and its application in the preparation of chiral omeprazole drugs.

[0006] This invention utilizes protein engineering techniques to further modify the protein molecularly, providing a thioether monooxygenase mutant with enhanced protein expression and significantly improved catalytic activity, stability, and substrate loading capacity for omeprazole thioether. The invention also includes a nucleic acid encoding the thioether monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a catalyst containing the recombinant thioether monooxygenase mutant, and the application of the recombinant thioether monooxygenase catalyst in the preparation of chiral proton pump inhibitors (PPIs).

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] One technical solution provided by this invention is a thioether monooxygenase mutant with significantly enhanced catalytic activity, expression level, stability, and substrate loading capacity. Using wild-type thioether monooxygenase (WT) with the amino acid sequence shown in SEQ ID No. 2, multiple thioether monooxygenase mutants with significantly enhanced protein expression, catalytic activity, stability, and substrate loading capacity were identified through a random mutagenesis method combined with high-throughput screening using an ELISA reader and further screening using HPLC shake flasks.

[0009] The thioether monooxygenase mutant provided by this invention is a derived protein with a new amino acid sequence formed by replacing one or more amino acid residues from the amino acid sequence shown in SEQ ID No. 2 at positions 17 (Gly), 56 (Ser), 58 (Ser), 70 (Glu), 83 (Ser), 123 (Asn), and 128 (Asn) with other amino acid residues; or,

[0010] A protein derived from a new amino acid sequence formed by replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 3 at positions 131 (Lys), 133 (Thr), 207 (Arg), 252 (Thr), 321 (Thr), 336 (Glu), 456 (His), 472 (Thr), 526 (Lys), and 531 (Lys) with other amino acid residues.

[0011] The expression level, oxidation activity against omeprazole thioether, and stability of the derived protein were all improved compared to the thioether monooxygenase corresponding to the amino acid sequence shown in SEQ ID No. 2. The expression level of the derived protein was significantly increased, and its oxidation activity against omeprazole thioether was significantly improved, while its stability was also improved.

[0012] Preferably, the amino acid sequence of the thioether monooxygenase mutant is one of the following:

[0013] (1) Replace Gly at position 17 of the amino acid sequence shown in SEQ ID No. 2 with Gln;

[0014] (2) Replace the 56th Ser in the amino acid sequence shown in SEQ ID No. 2 with Ala;

[0015] (3) Replace the 58th Ser in the amino acid sequence shown in SEQ ID No. 2 with Ala;

[0016] (4) Replace Glu at position 70 of the amino acid sequence shown in SEQ ID No. 2 with Tyr;

[0017] (5) Replace the 83rd Ser in the amino acid sequence shown in SEQ ID No. 2 with Gln;

[0018] (6) Replace the 123rd position Asn in the amino acid sequence shown in SEQ ID No.2 with Phe;

[0019] (7) Replace Asn at position 128 of the amino acid sequence shown in SEQ ID No. 2 with Thr;

[0020] (8) Replace Gly at position 17 of the amino acid sequence shown in SEQ ID No. 2 with Gln and Ser at position 56 with Ala;

[0021] (9) Replace the 58th Ser in the amino acid sequence shown in SEQ ID No.2 with Ala, and replace the 123rd Asn with Leu;

[0022] (10) Replace Glu at position 70 of the amino acid sequence shown in SEQ ID No. 2 with Tyr, and replace Ser at position 83 with Gln.

[0023] (11) Replace Gly at position 17 of the amino acid sequence shown in SEQ ID No.2 with Gln, Ser at position 56 with Ala, and Ser at position 58 with Ala;

[0024] (12) Replace Glu at position 70 of the amino acid sequence shown in SEQ ID No.2 with Tyr, replace Asn at position 123 with Leu, and replace Asn at position 128 with Thr;

[0025] (13) Replace Gly at position 17 with Gln, Glu at position 70 with Tyr, Asn at position 123 with Phe, and Thr at position 321 with Ser in the amino acid sequence shown in SEQ ID No.2;

[0026] (14) Replace Lys at position 131 of the amino acid sequence shown in SEQ ID No. 3 with Thr;

[0027] (15) Replace Thr at position 133 of the amino acid sequence shown in SEQ ID No. 3 with Ile;

[0028] (16) Replace Arg at position 207 of the amino acid sequence shown in SEQ ID No. 3 with Leu;

[0029] (17) Replace Thr at position 252 of the amino acid sequence shown in SEQ ID No. 3 with Asn;

[0030] (18) Replace Thr at position 321 of the amino acid sequence shown in SEQ ID No. 3 with Ser;

[0031] (19) Replace Glu at position 336 of the amino acid sequence shown in SEQ ID No. 3 with Lys;

[0032] (20) Replace His at position 456 of the amino acid sequence shown in SEQ ID No. 3 with Gln;

[0033] (21) Replace Thr at position 472 of the amino acid sequence shown in SEQ ID No.3 with Ser;

[0034] (22) Replace Lys at position 526 of the amino acid sequence shown in SEQ ID No.3 with Arg;

[0035] (23) Replace Lys at position 531 of the amino acid sequence shown in SEQ ID No.3 with His;

[0036] (24) Replace Thr at position 133 of the amino acid sequence shown in SEQ ID No.3 with Ile and Arg at position 207 with Leu;

[0037] (25) Replace Thr at position 252 of the amino acid sequence shown in SEQ ID No.3 with Asn and Thr at position 321 with Ser;

[0038] (26) Replace Glu at position 336 of the amino acid sequence shown in SEQ ID No.3 with Lys and His at position 456 with Gln;

[0039] (27) Replace Thr at position 472 of the amino acid sequence shown in SEQ ID No.3 with Ser, and replace Lys at position 526 with Gln;

[0040] (28) Replace Lys at position 131 with Thr, Thr at position 133 with Ile, and Arg at position 207 with Leu in the amino acid sequence shown in SEQ ID No.3;

[0041] (29) Replace the 252nd Thr with Asn, the 321st Thr with Ser, the 336th Glu with Lys, and the 472nd Thr with Ser in the amino acid sequence shown in SEQ ID No.3;

[0042] (30) Replace Lys at position 131 with Thr, Thr at position 252 with Asn, His at position 456 with Gln, and Thr at position 472 with Ser in the amino acid sequence shown in SEQ ID No.3.

[0043] (31) Replace Lys at position 131 with Thr, Thr at position 133 with Leu, Glu at position 336 with Lys, Lys at position 526 with Gln, His at position 456 with Gln, and Lys at position 531 with His.

[0044] (32) Replace Lys at position 131 with Thr, Thr at position 133 with Leu, Thr at position 321 with Ser, Glu at position 336 with Lys, Lys at position 526 with Gln, His at position 456 with Gln, and Lys at position 531 with His.

[0045] The second technical solution of the present invention provides a nucleic acid encoding the thioether monooxygenase mutant and a recombinant expression vector containing the encoding gene. The encoding gene expresses the thioether monooxygenase mutant as described in technical solution one, which is derived from cloning the gene sequences of the series of thioether monooxygenase mutants described in technical solution one using genetic engineering techniques. The recombinant expression vector is constructed by linking the nucleotide sequence of the thioether monooxygenase gene of the present invention to various commercially available empty vectors using conventional methods in the art. The commercially available empty vector can be any conventional plasmid vector in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the corresponding thioether monooxygenase mutant. Preferred plasmid vectors differ for different expression hosts. Those skilled in the art are generally aware of how to select appropriate vectors, promoters, enhancers, and host cells. For Pichia pastoris hosts, the preferred plasmid vector is pPIC9K. For example, the recombinant expression vector of Pichia pastoris described in this invention can be prepared by the following method: the DNA fragment of the thioether monooxygenase mutant gene sequence obtained by PCR amplification is digested with restriction endonucleases NdeI and Hind III, and the empty vector plasmid pPIC9K is also digested with restriction endonucleases NdeI and Hind III. The DNA fragment of the thioether monooxygenase mutant and the empty vector plasmid are recovered and ligated using T4 DNA ligase to construct a recombinant expression vector containing the nucleic acid molecule encoding the thioether monooxygenase for expression in Pichia pastoris.

[0046] The third technical solution of the present invention provides a recombinant expression transformant containing the thioether monooxygenase mutant gene of the present invention. The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into the corresponding host cell using conventional techniques in the art, provided that the recombinant co-expression vector can stably replicate spontaneously and that the encoded thioether monooxygenase gene can be effectively expressed. The host cell is preferably Pichia pastoris, more preferably protease-deficient Pichia pastoris SMD1168.

[0047] The fourth technical solution of the present invention provides a recombinant thioether monooxygenase mutant catalyst, wherein the recombinant thioether monooxygenase mutant catalyst is any one of the following forms:

[0048] (1) Cultivate and isolate the secretory supernatant of the recombinant expression transformant described in this invention;

[0049] (2) The crude enzyme powder is obtained by freeze-drying the secretory supernatant of the thioether monooxygenase mutant.

[0050] The culture methods and conditions for the recombinant expression transformants are conventional methods and conditions in the art. Different preferred culture methods and conditions are adopted for recombinant expression transformants constructed using different hosts, as long as the recombinant expression transformants can grow and efficiently produce the thioether monooxygenase mutant of the present invention.

[0051] For recombinant Pichia pastoris, the preferred culture medium is BMGY medium: 10 g / L glycerol, 20 g / L peptone, 10 g / L yeast extract, 40 mg / L biotin, 13.4 g / L amino acid-free yeast nitrogen source, and 100 mM potassium phosphate buffer, pH 6.0-6.5. The preferred culture method is as follows: the recombinant yeast is inoculated into BMGY medium containing ampicillin and cultured at 20-30°C. When the optical density OD of the culture medium... 600 When the pH reaches 1.3-2.0 (preferably 1.5), the culture medium is replaced with BMMY (10 mL / L methanol, 20 g / L peptone, 10 g / L yeast extract, 40 mg / L biotin, 13.4 g / L amino acid-free yeast nitrogen source, and 100 mM potassium phosphate buffer, pH 6.0). Pure methanol, equivalent to 1% of the culture medium volume, is added every 24 hours for induction, continuing induction for 72 hours to efficiently induce the recombinant Pichia pastoris to secrete and express the recombinant thioether monooxygenase mutant described in this invention. After cultivation, the culture medium is centrifuged at high speed, and the supernatant is collected to obtain the crude enzyme solution of the thioether monooxygenase mutant.

[0052] The collected crude enzyme solution was frozen at -80°C and then dried at low temperature using a vacuum freeze dryer to obtain lyophilized enzyme powder. The obtained lyophilized enzyme powder was stored in a refrigerator at 4°C for easy use.

[0053] The fifth technical solution of this invention provides a high-throughput screening method for the expression level of thioether monooxygenase mutant proteins. The principle of this high-throughput method is that after conjugating thioether monooxygenase to the fluorescent protein sfGFP, the fluorescence value increases with the increase of thioether monooxygenase expression. (See appendix) Figure 1 Specifically, epPCR was used to construct the target fragment, while the remaining fragments were constructed using conventional PCR. After successful construction, the fragments were transformed into the corresponding expression cells and screened for fluorescence using flow cytometry, followed by further screening using an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] The sixth technical solution of the present invention provides a high-throughput screening method for the enzyme activity of the thioether monooxygenase mutant, see appendix. Figure 2 Specifically, a colony-picking robot was used to pick the recombinant thioether monooxygenase transformants from the solid culture medium into 96-well plates (primary culture plates), and they were cultured overnight at 20-30°C and 800 rpm. Using an automated pipetting system, the transformants were transferred from the primary culture plates to secondary culture plates and cultured at 20-30°C and 800 rpm for 5-10 hours. The recombinant thioether monooxygenase mutants were then induced to express for 72-96 hours, followed by centrifugation to obtain the secretory supernatant. Add 0.2–1.0 mM omeprazole thioether as a screening substrate, 10–50 μL methanol as a co-solvent, and 50–90 μL potassium phosphate buffer containing NADPH (final concentration 0.2–1.0 mM). React at 25–35 °C and 800 rpm. After 2 hours, add 500 μL ethyl acetate and shake to extract. After centrifugation, take 100 μL of supernatant and mix it with 100 μL formic acid. Read the absorbance at 360 nm on a microplate reader. A higher absorbance indicates higher enzyme activity of the thioether monooxygenase mutant.

[0055] The seventh technical solution of the present invention provides a high-throughput screening method for methanol tolerance of the thioether monooxygenase mutant. Specifically, after obtaining the secretory supernatant as described in technical solution five of the present invention, it is co-incubated with 20% methanol for 5 hours. Subsequently, reactants are added as described in technical solution five for reaction and screening. The absorbance value is higher than that of the parent mutant, indicating that the methanol tolerance of the thioether monooxygenase mutant is improved.

[0056] The eighth technical solution of the present invention is to provide the application of the recombinant thioether monooxygenase mutant catalyst as described in technical solution one or the recombinant thioether monooxygenase mutant catalyst as described in technical solution four in the asymmetric oxidation of precatalytic chiral phase thioether substrates by coupling with a dehydrogenase.

[0057] In some embodiments of the present invention, the dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase or isopropanol dehydrogenase.

[0058] Furthermore, the dehydrogenase described in this invention is any one of the following dehydrogenases:

[0059] (1) Formate dehydrogenase FDH (Appl Biochem Biotech 2020, 192, 530–543), in the form of formate and NADP. + As a substrate, it catalyzes the oxidation of formate, while NADP... + Reducing to NADPH;

[0060] (2) Glucose dehydrogenase GDH (ChemBioChem 2020, 21:2680–2688), which dehydrogenases glucose and NADP. + As a substrate, it catalyzes the oxidation of glucose, while NADP... + Reducing to NADPH;

[0061] (3) Alcohol dehydrogenase ADH (Tetrahedron Lett 2021,84:153455), using short-chain alcohols and NADP. + As a substrate, it catalyzes the oxidation of short-chain alcohols, while NADP... + Restored to NADPH.

[0062] In some embodiments of the present invention, the enzymatic reaction is carried out in a buffer solution with a pH of 6.0–10.0 at 25–40°C. The reaction system includes omeprazole sulfide at a final concentration of 10–30 g / L, 5–25% (v / v) methanol as a co-solvent, 10–300 mM sodium formate, and 0–1 mM NADP. + 10-800 U / L as described in technical solution four, recombinant thioether monooxygenase mutant catalyst.

[0063] Preferably, the reaction is carried out under aeration and stirring, with an aeration rate of 0–2 vvm, and the reaction time is determined by the complete conversion of the substrate or the product concentration no longer increasing.

[0064] During the reaction intervals, a 0.5 mL sample of the reaction solution was taken, and 0.5 mL of ethyl acetate was added for extraction. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 0.3 mL of ethanol was added to dissolve the extract. The solution was filtered through a 0.22 μm pore size membrane and subjected to liquid chromatography analysis to determine the substrate conversion rate and the ee value of the product. Specific analytical conditions are as follows:

[0065] The chromatographic column was a Daicel Chiralpak IA, the mobile phase was n-heptane:ethanol = 70:30 (v / v), and the flow rate was 0.5 mL / min. -1 The column temperature was 40℃, and the ultraviolet detection wavelength was 300nm.

[0066] Compared with the prior art, the technical effects of the present invention are mainly reflected in the following aspects:

[0067] Compared with other biocatalysts for preparing optically pure proton pump inhibitors, the thioether monooxygenase mutant provided by this invention has the advantages of simple preparation, high expression level, high catalytic activity, good stability, and high substrate loading capacity, showing broad application prospects in industrial applications. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of high-throughput screening of enzyme expression levels in this invention.

[0069] Figure 2 This is a schematic diagram of the high-throughput screening process for viability and stability in this invention.

[0070] Figure 3 This is a schematic diagram of the construction of the random mutation library in this invention.

[0071] Figure 4 This is a schematic diagram of the reaction process of the asymmetric oxidation of pre-chiral prazole thioether catalyzed by the thioether monooxygenase mutant in this invention. Detailed Implementation

[0072] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0073] The reaction or detection conditions described in this invention can be combined or modified based on common knowledge in the art, and can be verified experimentally. The technical solutions and effects of this invention will be clearly and completely described below with reference to specific embodiments. However, the scope of protection of this invention is not limited to these embodiments; all changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0074] The plasmid vector pPIC9K was purchased from Novagen.

[0075] The protease-deficient Pichia pastoris SMD1168 competent cells, 2×Taq PCR MasterMix, and agarose gel DNA recovery kit were all purchased from Beijing Tiangen Biotech Co., Ltd.

[0076] The restriction endonucleases Nde I and Hind III are both commercially available products from New England Biolabs (NEB).

[0077] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the product instructions for the kit.

[0078] The culture media and detection methods involved in the following examples are as follows:

[0079] YPD medium: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L anhydrous glucose dissolved in deionized water.

[0080] BMGY culture medium: 15 g / L tryptone, 10 g / L yeast extract and 10 g / L glycerol are dissolved in deionized water, sterilized and cooled, and then the mother liquor is added to make the system reach 1×YNB nutrient salts.

[0081] The chiral HPLC analysis method is as follows:

[0082] Instrument: Shimadzu HPLC 2010A; Column: Chiralpak IA; Mobile phase: n-heptane:ethanol = 70:30 (v / v); Flow rate: 0.6 mL / min -1 Column temperature: 40℃; injection volume: 10μL; detector: UV detector; detection wavelength: 300nm.

[0083] Example 1: Construction of a random mutant library of thioether monooxygenase (AcPSMO)

[0084] The amino acid sequence of thioether monooxygenase (AcPSMO) is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding thioether monooxygenase (AcPSMO) is shown in SEQ ID No. 1.

[0085] Based on the open reading frame of AcPSMO, the upstream and downstream primers are designed as follows:

[0086] The upstream primer is shown in SEQ ID No. 4.

[0087] Downstream primer, as shown in SEQ ID No. 5.

[0088] To shorten library construction time and maximize library capacity, the process of extracting plasmids from *E. coli* and linearizing them for homologous recombination in the *Pichia pastoris* genome to achieve complete expression frames of target genes was omitted. Instead, a PCR-dependent strategy was used to construct a *Pichia pastoris* error-prone mutant library. First, error-prone PCR and conventional vector reverse PCR were used to amplify homologous fragments containing vectors "a" and "b" (as shown in the attached image). Figure 3As shown, three gene fragments of different sizes (error-prone gene fragment, 3.5K long, and 3.5K short) with homologous fragments "a" and "b" each 60 bp in length were mixed according to an optimized molar ratio (error-prone gene fragment: vector fragment = 10:1) and directly transformed into Pichia pastoris by electroporation. Homologous recombination of the three gene fragments of different sizes carrying the homologous fragments "a" and "b" within the Pichia pastoris cell forms a complete gene expression framework. Finally, homologous recombination is performed in the histidine auxotrophic Pichia pastoris genome using the PpHIS4 site, integrating the expression cassette into the Pichia pastoris genome, thus achieving the integration of the complete foreign gene "reading frame" into the genome, enabling successful expression of the target gene. A schematic diagram of the entire process is attached. Figure 3 .

[0089] Using the constructed plasmid pPIC3.5K-AcPSMO-sfGFP as the parent, PCR of the target gene was performed using an error-prone PCR kit, while the remaining vector components were processed using conventional PCR. Adding an optimized Mn2+ concentration of 80 μM controlled the mutation rate of the target error-prone PCR fragment to 1-2 bases, maximizing the preservation of the target protein's enzymatic properties. After error-prone PCR of the target gene, conventional PCR of the vector was performed.

[0090] After all PCR was completed, the fragments were recovered using a gel purification kit (ddH2O recovery). The fragments were mixed at a molar ratio of 10:1 and then introduced into Pichia pastoris using electroporation. After 2 hours of recovery, the fragments were plated on MD plates and incubated at 30°C for 48 hours. Dense milky white colonies appeared, indicating that the Pichia pastoris error-prone mutant library was successfully constructed.

[0091] Example 2: Flow cytometry screening of mutants with increased expression of thioether monooxygenase (AcPSMO) in Pichia pastoris

[0092] Pretreatment before flow cytometry sorting: The colony mutant library from Minimal Dextrose (MD) plates was resuspended in sterile deionized water and aliquoted into 2 mL Eppendorf tubes. After centrifugation at 12,000 rpm for 2 min, the supernatant was discarded. The cells were then fully resuspended again in 1.5 mL of sterile BMMY medium containing 100 μg / mL ampicillin. The cells were incubated at 30°C and 220 rpm for 36 h, followed by centrifugation at 13,000 rpm for 3 min and discarding the supernatant. The cells were immediately resuspended in 50 mM, pH 6.0 PBS buffer, then filtered through a 40 μm sterile cell filter to separate single cells. Finally, PBS buffer was added to adjust the OD of the final bacterial count. 600The temperature range is 2-4°C. Resuspended bacterial cells can be directly sorted by flow cytometry or temporarily stored at 4°C.

[0093] Flow cytometry cell sorting: Before sorting, turn on the air compressor, replace the collection nozzle in the sorter, insert the 100μm yeast sorting chip, and select the 488nm excitation light channel. The instrument will then automatically complete the optical path calibration and collection calibration. After completion, load the yeast sample to be sorted into a dedicated 10mL sorting tube, and simultaneously insert a 10mL collection tube into the instrument's collection port to collect cells with high fluorescence intensity. Turn on the flow cytometry switch, change the Events setting to fluctuate with fluorescence intensity, and adjust the collection rate to approximately 8,000-10,000 events / s. Finally, based on the fluorescence response map provided by the software, directly select the sorting valves "A" and "B" in the map and click "sort star" to start sorting. The sorted cells can be sorted twice to obtain strains with even higher fluorescence intensity.

[0094] Through screening, mutants with significantly increased enzyme expression were obtained, and the protein expression levels of these mutants were then quantified. A series of mutants with increased protein expression were selected, and the sequences and protein expression levels of these mutants are listed in Table 1.

[0095] Table 1: List of AcPSMO mutant sequences and corresponding protein expression modifications for thioether monooxygenase

[0096]

[0097]

[0098] Example 3: Screening for AcPSMO mutants with increased omeprazole thioetherase activity

[0099] Transformants from transformation plates were transferred to 96-well deep-well plates using a Qpix 450 automated monoclonal sorting system from Molecular Devices (Shanghai) Co., Ltd., and cultured overnight at 20-30°C and 800 rpm. Using a TECAN Freedom EVO fully automated liquid handling workstation, 50 μL of bacterial culture was transferred from the primary plate wells to the corresponding secondary plate wells and cultured at 20-30°C and 800 rpm for 5-10 h, followed by induction culture at 20-30°C for 72 h. The culture was then centrifuged at 3500 × g for 10 min at 4°C, and the supernatant was collected. High-throughput screening of expressed proteins was performed in 96-well plates. Mutants with high omeprazole thioether activity were purified and characterized, and the corresponding genes were sequenced.

[0100] The high-throughput activity screening assay for the AcPSMO mutant is as follows: Potassium phosphate buffer (100mM, pH 9.0) containing 1mM substrate omeprazole sulfide, 1mM coenzyme NADPH, 100mM cosubstrate, and a certain amount of dehydrogenase powder are dispensed into 96-well plates. The plates are preheated to 30°C, and then an appropriate amount of AcPSMO mutant secretion supernatant is added. The plates are incubated at 30°C with shaking. The absorbance change of NADPH at 360nm is detected using a microplate reader, and the absorbance change value within 1 minute is recorded. The corresponding enzyme activity is then calculated.

[0101] Example 4: Screening for AcPSMO mutants with enhanced methanol tolerance

[0102] Due to the poor solubility of the substrate, precipitation occurs when the substrate concentration is increased. To improve substrate loading, the concentration of the cosolvent can be increased; however, high concentrations of cosolvent can rapidly inactivate the enzyme. To address this issue, a random mutagenesis method was used to screen for AcPSMO mutants with enhanced methanol tolerance.

[0103] By making minor modifications to the procedures in Example 3, the AcPSMO mutant with improved methanol tolerance can be screened. Specifically, the yeast secretion supernatant obtained in Example 1 was co-incubated with 20% methanol for 5 hours before the reaction was carried out. The absorbance value was higher than that of the wild type, indicating that the residual enzyme activity was higher, meaning that the AcPSMO mutant had improved methanol tolerance compared to the wild type.

[0104] Example 5: Combined Mutations of Beneficial AcPSMO Mutants

[0105] Based on the mutations in Examples 3 and 4, several dominant mutants with good activity and methanol tolerance were obtained. Site-directed mutagenesis was used to combine the relevant mutation sites involved in the obtained dominant mutants. PCR system (25 μL): 15 ng AcPSMO plasmid, 1.5 μL each of upstream and downstream primers (10 μM), 12.5 μL Prime Star DNA polymerase, 0.5 μL DMSO, and sterile water to make up to 25 μL. PCR reaction program: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 30 s; (3) 60℃ annealing for 30 s; (4) 72℃ extension for 100 s; steps (2) to (4) were performed for a total of 15 cycles; finally, the extension was performed at 72℃ for 10 min, and the product was stored at 4℃. After obtaining the PCR product, it was digested with Dpn I at 37℃ for 1 h and then used for transformation.

[0106] Through screening, mutants with significantly enhanced activity were obtained. The methanol tolerance of these mutants was then characterized, and a series of mutants with increased methanol tolerance were selected as the preferred mutants. The sequences, activities, and stability of these mutants are listed in Table 2. In Table 2, the sequence numbers correspond to a series of sequences listed later in the table.

[0107] Table 2: List of AcPSMO mutant sequences and corresponding activity improvements for thioether monooxygenase

[0108]

[0109]

[0110] Example 6: Determination of the activity of omeprazole thioether by the thioether monooxygenase mutant.

[0111] The enzyme activity of AcPSMO on omeprazole thioether substrate was determined using the following steps: 0.2 mM omeprazole thioether substrate and 0.2 mM NADPH were added to 0.5 mL of the reaction system (0.1 M KPB buffer, pH 9.0). After incubation at 30 °C for 2 min, an appropriate amount of purified enzyme solution was added and quickly mixed. The reaction was carried out at 30 °C and 1000 rpm for 10 min. The reaction was terminated with 0.5 mL of ethyl acetate and extracted. The supernatant was centrifuged and filtered through a 0.22 μm pore size filter membrane for liquid chromatography analysis to determine the substrate conversion rate and the ee of the product.

[0112] The specific analytical conditions for substrate conversion and ee are as follows:

[0113] Instrument: Shimadzu HPLC 2010A; Column: Chiralpak IA; Mobile phase: n-heptane:ethanol = 70:30 (v / v); Flow rate: 0.5 mL / min -1 Column temperature: 40℃; injection volume: 10μL; detector: UV detector; detection wavelength: 300nm.

[0114] Enzyme activity unit (U) is defined as: the amount of enzyme required per minute to catalyze the conversion of 1 μmol of omeprazole thioether substrate into (S)-omeprazole under the above conditions.

[0115] Example 7 Expression of thioether monooxygenase mutant

[0116] The mutant expression strain was inoculated into YPD liquid medium (peptone: 20 g / L, yeast extract: 10 g / L, glucose: 20 g / L) containing 100 μg / mL bleomycin and cultured at 30℃ with shaking at 250 rpm for 24 h. Then, 1% of the culture was inoculated into 100 ml of BMGY liquid medium (peptone: 20 g / L, yeast extract: 10 g / L, glycerol: 10 g / L, amino acid-free yeast nitrogen source (YNB, BD Difco): 13.6 g / L, biotin: 0.4 mg / L, final concentration 200 mM potassium phosphate buffer, pH 6.0) containing 100 μg / mL ampicillin and cultured at 30℃ with shaking at 250 rpm. The culture was then incubated until the turbidity OD of the culture medium was measured. 600 When the culture reaches 1.5, stop the culture, centrifuge to collect yeast cells, carefully decan the BMGY medium supernatant, and then resuspend the collected cells in 100 ml of BMGY medium (methanol: 10 ml / L, peptone: 20 g / L, yeast extract: 10 g / L, biotin: 0.4 mg / L, amino acid-free yeast nitrogen source 13.6 g / L, final concentration 100 mM potassium phosphate buffer, pH 6.0). Continue culturing at 30°C and 250 rpm in a shaker, adding 1.5 ml of pure methanol every 24 h for induction, continuing the induction process for 72 h. After the culture is complete, centrifuge the culture at 4°C and 8000 × g to remove the cells, obtaining the yeast secretion supernatant.

[0117] Example 8: Preparation of AcPSMO lyophilized enzyme powder

[0118] The crude enzyme solution from the secretory supernatant collected in Example 7 was frozen at -80°C and then dried at low temperature using a vacuum freeze dryer to obtain lyophilized enzyme powder. The obtained lyophilized enzyme powder can be conveniently used by storing it in a refrigerator at 4°C.

[0119] Example 9 AcPSMO M21 (Shown SEQ ID No. 3) Asymmetric oxidation of 10 g / L omeprazole thioether catalyzed by secretory supernatant or lyophilized enzyme powder.

[0120] The reaction process of the asymmetric oxidation of pre-chiral p-prazole thioether and the chiral sulfoxide peroxidation catalyzed by the thioether monooxygenase mutant is shown in the attached figure. Figure 4 As shown.

[0121] Add AcPSMO to a 100 mL reaction system M21 Add 40 mL of secretory supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 18 U of formate dehydrogenase powder, and then add omeprazole sulfide, methanol, sodium formate, and NADP to the above system. +The final concentrations were 10 g / L, 10% (v / v), 100 mM, and 0.2 mM, respectively. The reaction was carried out with shaking at 30 °C and 200 rpm. 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 12 h of reaction, the substrate conversion was greater than 99%, and the product ee value was greater than 99% (S).

[0122] Example 10 AcPSMO M27 (Mutant M27 in Table 2) Asymmetric oxidation of 20 g / L omeprazole thioether catalyzed by secretory supernatant or lyophilized enzyme powder.

[0123] Add AcPSMO to a 100 mL reaction system. M27 Add 35 mL of secretion supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 25 U of formate dehydrogenase powder, and then add omeprazole sulfide, methanol, sodium formate, and NADP to the above system. + The final concentrations were 20 g / L, 15% (v / v), 120 mM, and 0.2 mM, respectively. The reaction was carried out with shaking at 30 °C and 200 rpm. 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 15 h of reaction, the substrate conversion was greater than 97%, and the product ee value was greater than 99% (S).

[0124] Example 11 AcPSMO M35 (Mutant M35 in Table 2) Asymmetric oxidation of 30 g / L omeprazole thioether catalyzed by secretory supernatant or lyophilized enzyme powder.

[0125] Add AcPSMO to a 100 mL reaction system M35 Add 30 mL of secretion supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 30 U of formate dehydrogenase powder, and then add omeprazole sulfide, methanol, sodium formate, and NADP to the above system. +The final concentrations were 30 g / L, 20% (v / v), 150 mM, and 0.2 mM, respectively. The reaction was carried out with shaking at 30 °C and 200 rpm. 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 18 h of reaction, the substrate conversion was greater than 98.4%, and the product ee value was greater than 99% (S).

[0126] Example 12 AcPSMO M39 (Mutant numbered M39 in Table 2) Asymmetric oxidation of 40 g / L omeprazole thioether catalyzed by secretory supernatant or lyophilized enzyme powder.

[0127] Add AcPSMO to a 100 mL reaction system M39 Add 25 mL of secretory supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 40 U of formate dehydrogenase powder, and then add omeprazole sulfide, methanol, sodium formate, and NADP to the above system. + The final concentrations were 40 g / L, 25% (v / v), 200 mM, and 0.2 mM, respectively. The reaction was carried out with shaking at 30 °C and 200 rpm. 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 24 h of reaction, the substrate conversion was greater than 92.4%, and the product ee value was greater than 99% (S).

[0128] Example 13 AcPSMO M39 (Mutant number M39 in Table 2) secretory supernatant or lyophilized enzyme powder coupled with isopropanol dehydrogenase catalyzes asymmetric oxidation of 40 g / L omeprazole thioether.

[0129] Add AcPSMO to a 100 mL reaction system M39 Add 25 mL of secretory supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 40 U of isopropanol dehydrogenase powder, and then add omeprazole sulfide, isopropanol, and NADP to the above system. +The final concentrations were 40 g / L, 25% (v / v), and 0.2 mM, respectively. The reaction was carried out with shaking at 30 °C and 200 rpm. 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 24 h of reaction, the substrate conversion was greater than 91.3%, and the product ee value was greater than 99% (S).

[0130] Example 14 AcPSMO M39 Secretory supernatant or lyophilized enzyme powder coupled with glucose dehydrogenase catalyzes asymmetric oxidation of 40 g / L omeprazole thioether.

[0131] Add AcPSMO to a 100 mL reaction system M39 Add 25 mL of secretory supernatant or a certain amount of lyophilized enzyme powder from Example 8 (adjust pH to 8.0), 40 U of glucose dehydrogenase powder, and then add omeprazole sulfide, methanol, glucose, and NADP to the above system. + The final concentrations were 40 g / L, 25% (v / v), 200 mM, and 0.2 mM, respectively. The reaction was carried out at 30°C and 200 rpm with shaking. During the reaction, the pH of the reaction system was controlled at approximately 8, and 0.5 mL of the reaction solution was intermittently sampled and extracted with 0.5 mL of ethyl acetate. 0.3 mL of the extract was dried over anhydrous sodium sulfate, and the ethyl acetate was evaporated. Then, 1 mL of ethanol was added to dissolve the extract. The substrate conversion and product ee were determined by HPLC analysis as described in Example 6. After 24 h of reaction, the substrate conversion was greater than 90.2%, and the product ee value was greater than 99% (S).

[0132] The sequences involved in this invention are as follows:

[0133] The nucleotide sequence of the gene encoding thioether monooxygenase (AcPSMO) (SEQ ID NO.1)

[0134]

[0135]

[0136] The amino acid sequence of thioether monooxygenase (AcPSMO) (SEQ ID NO.2)

[0137]

[0138] Mutant AcPSMO M21 The amino acid sequence (SEQ ID NO.3)

[0139]

[0140]

[0141] Upstream primer (SEQ ID NO.4)

[0142]

[0143] Downstream primer (SEQ ID NO.5)

[0144]

[0145] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A mutant sulfide monooxygenase enzyme characterized in that, The thioether monooxygenase mutant is a derivative protein shown in any one of the following amino acid sequences: (1) substituting Gly at position 17 for Gin of the amino acid sequence shown in SEQ ID No. 2; (2) substituting Gly at position 17 for Gin and Ser at position 56 for Ala of the amino acid sequence shown in SEQ ID No. 2; (3) substituting Gly at position 17 for Gin, Ser at position 56 for Ala and Ser at position 58 for Ala of the amino acid sequence shown in SEQ ID No. 2; (4) substituting Gly at position 17 for Gin, Glu at position 70 for Tyr, Asn at position 123 for Phe and Thr at position 321 for Ser of the amino acid sequence shown in SEQ ID No. 2; (5) substituting Lys at position 131 for Thr of the amino acid sequence shown in SEQ ID No. 3; (6) substituting Thr at position 133 for Ile of the amino acid sequence shown in SEQ ID No. 3; (7) substituting Arg at position 207 for Leu of the amino acid sequence shown in SEQ ID No. 3; (8) substituting Thr at position 252 for Asn of the amino acid sequence shown in SEQ ID No. 3; (9) substituting Thr at position 321 for Ser of the amino acid sequence shown in SEQ ID No. 3; (10) substituting Glu at position 336 for Lys of the amino acid sequence shown in SEQ ID No. 3; (11) substituting His at position 456 for Gin of the amino acid sequence shown in SEQ ID No. 3; (12) substituting Thr at position 472 for Ser of the amino acid sequence shown in SEQ ID No. 3; (13) substituting Lys at position 526 for Arg of the amino acid sequence shown in SEQ ID No. 3; (14) substituting Lys at position 531 for His of the amino acid sequence shown in SEQ ID No. 3; (15) substituting Thr at position 133 for Ile and Arg at position 207 for Leu of the amino acid sequence shown in SEQ ID No. 3; (16) substituting Thr at position 252 for Asn and Thr at position 321 for Ser of the amino acid sequence shown in SEQ ID No. 3; (17) substituting Glu at position 336 for Lys and His at position 456 for Gin of the amino acid sequence shown in SEQ ID No. 3; (18) substituting Thr at position 472 for Ser and Lys at position 526 for Gin of the amino acid sequence shown in SEQ ID No. 3; (19) substituting Lys at position 131 for Thr, Thr at position 133 for Ile and Arg at position 207 for Leu of the amino acid sequence shown in SEQ ID No. 3; (20) replacing Thr at position 252, Thr at position 321, Glu at position 336, and Thr at position 472 in the amino acid sequence shown in SEQ ID No. 3 with Asn, Ser, Lys, and Ser, respectively; (21) replacing Lys at position 131, Thr at position 252, His at position 456, and Thr at position 472 in the amino acid sequence shown in SEQ ID No. 3 with Thr, Asn, Gln, and Ser, respectively; (22) replacing Lys at position 131, Thr at position 133, Glu at position 336, Lys at position 526, His at position 456, and Lys at position 531 in the amino acid sequence shown in SEQ ID No. 3 with Thr, Leu, Lys, Gln, Gln, and His, respectively; (23) replacing Lys at position 131, Thr at position 133, Thr at position 321, Glu at position 336, Lys at position 526, His at position 456, and Lys at position 531 in the amino acid sequence shown in SEQ ID No. 3 with Thr, Leu, Ser, Lys, Gln, Gln, and His, respectively.

2. An isolated nucleic acid, comprising, The nucleic acid encodes any one of the thioether monooxygenase mutants as claimed in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as claimed in claim 2.

4. A recombinant expression transformant, characterized by, The recombinant expression transformant comprises the expression vector as claimed in claim 3.

5. A recombinant sulfϊde monooxygenase mutant catalyst characterized in that, The recombinant thioether monooxygenase mutant catalyst is in any one of the following forms: (1) culturing and isolating the secreted supernatant of the recombinant expression transformant as claimed in claim 4; (2) freeze-drying the secreted supernatant of the thioether monooxygenase mutant to obtain a crude enzyme powder.

6. Use of the thioether monooxygenase mutant as claimed in claim 1 or the recombinant thioether monooxygenase mutant catalyst as claimed in claim 5 in the preparation of a chiral lansoprazole drug by coupling a dehydrogenase in the asymmetric oxidation of a chiral lansoprazole thioether substrate.

7. Use according to claim 6, wherein Using the recombinant thioether monooxygenase mutant to catalyze the asymmetric oxidation of omeprazole thioether as a substrate to synthesize (S)-omeprazole.

8. Use according to claim 7, wherein the compound is ###0002### During the reaction, coenzyme NADPH is oxidized to NADP+, and a dehydrogenase is used to catalyze the reduction of NADP+ to regenerate NADPH; the dehydrogenase is any one of formate dehydrogenase, glucose dehydrogenase, or isopropanol dehydrogenase.

9. The use according to claim 7, wherein the compound is ###0005### The reaction conditions are as follows: the concentration of the substrate omeprazole thioether is 10-40 g / L, the concentration of the cosolvent is 10%-25% (v / v), the molar ratio of sodium formate to the substrate is 1.0-2.0, the NADP+ addition amount is 0.05-2.0 mmol / L, the pH is 6.0-9.0, and the temperature is 20-40°C.

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