WO2020108462A1 - 一种单加氧酶及其在制备光学纯亚砜中的应用 - Google Patents

一种单加氧酶及其在制备光学纯亚砜中的应用 Download PDF

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WO2020108462A1
WO2020108462A1 PCT/CN2019/120798 CN2019120798W WO2020108462A1 WO 2020108462 A1 WO2020108462 A1 WO 2020108462A1 CN 2019120798 W CN2019120798 W CN 2019120798W WO 2020108462 A1 WO2020108462 A1 WO 2020108462A1
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张龑
郁惠蕾
许建和
吴殷琦
赵骞
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华东理工大学
江苏奥赛康药业有限公司
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Priority to CN201980059937.7A priority patent/CN113195518A/zh
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    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
    • C12Y114/14001—Unspecific monooxygenase (1.14.14.1)
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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Definitions

  • the invention relates to an unnatural thioether monooxygenase and the application of enzymatically catalyzed asymmetric oxidation of thioether compounds to prepare sulfoxide compounds.
  • Esomeprazole also known as (S)-omeprazole, has a chemical name of 5-methoxy-2-((S)-((4-methoxy-3,5-dimethyl- 2-pyridyl)methyl)sulfinyl)-1H-benzimidazole, the chemical structure is shown in formula I.
  • Esomeprazole is the first (S)-single configuration isomer of omeprazole, a proton pump inhibitor used clinically. This medicine is mainly used to treat duodenal ulcer, gastric ulcer, gastritis and gastrointestinal esophagitis. It has been clinically proven that the drug has lower toxic and side effects than racemate and (R)-omeprazole, and the efficacy is better.
  • Chemical methods use metal catalysts to synthesize omeprazole by asymmetric oxidation of thioether, but such methods have the disadvantages of limited optical purity, excessive oxidation, many by-products, and complicated separation and purification processes.
  • the enzymatic method is used to asymmetrically oxidize the sulfide compound to obtain a single enantiomer of chiral sulfoxide.
  • This method has less environmental pollution, fewer by-products, good atom economy, and high optical purity of the product, so the enzymatic method is used.
  • Asymmetric oxidation synthesis of chiral sulfoxide has received more and more attention.
  • the inventor has also screened and obtained a strain of Rhodococcus (CGMCC2547) that can catalyze the asymmetric oxidation of a series of latent chiral phenylalkyl sulfides and their derivatives, and obtained optically active chiral benzyl sulfoxide and its derivatives ( CN101372676B).
  • CGMCC2547 Rhodococcus
  • Czech scientists screened and obtained a strain of Bacillus pumilus Lysinibacillus sp., using its growth cells to catalyze the production of omeprazole sulfide (S)-omeprazole, but when the substrate concentration is only 0.1g/L, 44h The conversion rate is only 43% (Bioresources Technology 2011, 102:7621–7626).
  • Patent WO2011071982 carried out directed evolution of cyclohexanone monooxygenase NCIMB9871.
  • the engineered enzyme catalyzes the loading of 100g/L substrate, supplementing 10g/L of lyophilized enzyme powder in 5 batches, and the substrate is transformed after 69h of reaction.
  • the ee value reaches 99%.
  • the patent CN108118035A discloses the mutation of the 157th serine of the mutant 157 to asparagine, and the 435th serine to threonine.
  • the obtained catalytic substrate concentration of the mutant is as high as 165g/ L.
  • the present invention provides a monooxygenase.
  • the monooxygenase includes a mutant of the amino acid sequence shown in SEQ ID NO: 2, and the mutation mode of the mutant includes substitution of an amino acid residue at a specified position.
  • the designated positions include Xaa21, Xaa40, Xaa55, Xaa70, Xaa143, Xaa145, Xaa156, Xaa185, Xaa220, Xaa244, Xaa246, Xaa248, Xaa249, Xaa277, Xaa281, Xaa326, Xaa386, Xaa388, Xaa390 including the amino acid sequence shown in SEQ ID NO: 2 , Xaa405, Xaa426, Xaa430, Xaa432, Xaa433, Xaa435, Xaa438, Xa465, Xaa468, Xaa
  • the substitution of the amino acid residue at the designated position includes at least two substitutions of the amino acid residue at the designated position.
  • the monooxygenase may optionally include any one or more of the following mutation methods, and the mutation methods include:
  • the substitution of the amino acid residue at the specified position includes any one or several of the following substitution modes:
  • the substitution of the amino acid residue at the specified position includes any of the following substitution methods:
  • the present invention also provides a monooxygenase, the amino acid sequence of the monooxygenase: as shown in SEQ ID NO: 4, or as shown in SEQ ID NO: 6, or as shown in SEQ ID NO: 8 , Or as shown in SEQ ID NO: 10, or as shown in SEQ ID NO: 12, or as shown in SEQ ID NO: 14, or as shown in SEQ ID NO: 16, or as shown in SEQ ID NO: 18 , Or as shown in SEQ ID NO: 20, or as shown in SEQ ID NO: 22, or as shown in SEQ ID NO: 24, or as shown in SEQ ID NO: 26, or as shown in SEQ ID NO: 28 , Or as shown in SEQ ID NO: 30, or as shown in SEQ ID NO: 32, or as shown in SEQ ID NO: 34, or as shown in SEQ ID NO: 36, or as shown in SEQ ID NO: 38 , Or as shown in SEQ ID NO: 40, or as shown in SEQ ID NO: 42, or as shown in SEQ ID NO: 44, or as shown in
  • the present invention also provides an isolated nucleic acid encoding the above-mentioned monooxygenase.
  • the present invention also provides a recombinant expression vector, which contains the nucleic acid as described above.
  • the present invention is a recombinant expression transformant.
  • the recombinant expression transformant comprises a recombinant expression vector as described above.
  • the invention also provides a method for preparing the monooxygenase as described above, comprising the steps of: cultivating the recombinant expression transformant as described above, and isolating the monooxygenase therefrom.
  • the invention also provides an application of the monooxygenase as described above in the asymmetric catalytic oxidation of a latent chiral sulfide compound to a sulfoxide compound.
  • the latent chiral sulfide compound is selected from compounds represented by any one of the following chemical formulas:
  • Xaa number represents the amino acid residue at the "number” position in the amino acid sequence
  • Xaa40 represents the amino acid residue at the "40" position in the amino acid sequence.
  • amino acid residue (a) + Xaa number means that the amino acid residue at the "number" position in the amino acid sequence is “amino acid residue (a)”, which may also be abbreviated as “amino acid residue (a) + number” ";
  • amino acid residue (a) + number means that the amino acid residue at position "326" in the amino acid sequence is "K” (lysine).
  • amino acid residue (a) + Xaa number + amino acid residue (b) means that the amino acid residue at the "number" position in the amino acid sequence is replaced by "amino acid residue (a)” with “amino acid residue ( b)", which can also be abbreviated as “amino acid residue (a) + number + amino acid residue (b)”; for example, “K326C” means that the amino acid residue at position "326" in the amino acid sequence is composed of "K” (lysine Acid) with "C” (cysteine).
  • Xaa number refers to the amino acid position in SEQ ID NO: 2 as a reference.
  • the present invention analyzes the crystal structure of Acinetobacter thioether monooxygenase, establishes a high-throughput flat transparent circle screening method, and combines rational design and high-throughput screening to obtain the monooxygenase
  • the catalytic activity is high, and the reaction is added as a small amount of catalyst, which significantly improves the catalytic activity of the enzyme and the space-time yield of the catalytic reaction.
  • the scale of the reaction is not limited to the laboratory scale, providing a new organism for the industrial synthesis of chiral sulfoxide drugs. Catalyst resources.
  • the present invention uses the monooxygenase of the present invention to prepare chiral sulfoxide drugs with mild reaction conditions, environmental friendliness, high yield, high product optical purity, and few peroxide products And other advantages, it has a good industrial application prospect in the production of proton pump inhibitors for the treatment of gastric ulcer.
  • Figure 1 is a construction diagram of the recombinant plasmid pET28a-AcPSMO
  • Figure 2 is a diagram of the substrate channel of the single oxygenase AcPSMO and its key sites
  • Figure 3 is a diagram of the key sites affecting the production of by-products in the structure of the monooxygenase AcPSMO;
  • Figure 4 is a crystalline grade purified SDS-PAGE diagram, where lane 1 is the crushing liquid, 2 is the Ni eluent, 3 is the sample before molecular sieve loading, and 4-12 is the sample after molecular sieve separation;
  • Fig. 5 is a construction diagram of the flat panel transparent circle screening method, in which "+" and "-" in the left picture represent AcPSMO mutant and AcPSMO (SEQ ID NO: 2), respectively, and the right picture shows the screening of the actual mutant library.
  • the present invention analyzes and predicts a single oxygenase gene that may have oxidizing activity on sulfide through a bioinformatics method, successfully clones and expresses multiple single oxygenase genes, and verifies their functions; some of the verification results are shown in Table 1 As shown.
  • thioether mono Oxygenase can efficiently catalyze the oxidation of anisole (a compound of formula IIa) to benzosulfoxide (a compound of formula IIb).
  • the thioether monooxygenase is named AcPSMO, and its amino acid sequence is shown in SEQ ID NO: 2 .
  • the Acinetobacter calcoaceticus used in the present invention is deposited in the General Microbiological Center (CGMCC, No. 3, No. 1 Hospital, Beichen West Road, Chaoyang District, Beijing), China It is: March 20, 2014, and its deposit number is: CGMCC No. 8936.
  • CGMCC General Microbiological Center
  • Acinetobacter represents the genus name
  • calcoaceticus represents the species name
  • sp. represents the undetermined species name.
  • the inventor further identified Acinetobacter (Acinetobacter sp.) in the biological deposit certificate to the clock. It is Acinetobacter calcoaceticus.
  • the naturally-occurring monooxygenase AcPSMO catalyzes oxygenation to produce certain sulfoxide compounds with low catalytic activity, such as the oxidation of omeprazole sulfide (compound of formula IIIa) to the compound of formula IIIb, etc.
  • the present invention analyzes the monooxygenase
  • the crystal structure of AcPSMO has creatively found that the amino acid residues at certain positions are the key to influence the substrate to enter the catalytic center and affect the catalytic activity. Through a lot of research, a single oxygenase has been provided.
  • the amino acid sequence of the enzyme is obtained from the amino acid sequence shown in SEQ ID NO: 2 by including any two or more places that replace the amino acid residue at the specified position; for example, it can be any two places by replacing the amino acid residue at the specified position Offices, 3 Offices, 4 Offices, 5 Offices, 6 Offices, 7 Offices, 8 Offices, 9 Offices, 10 Offices, 11 Offices, 12 Offices, 13 Offices, 14 Offices, 15 Offices, 16 Offices, 17 Offices, 18 Offices, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.
  • the amino acid residue at the designated position is selected from: Xaa21, Xaa40, Xaa55, Xaa70, Xaa143, Xaa145, Xaa156, Xaa185, Xaa220, Xaa244, Xaa246, Xaa248, Xaa249, Xaa277, Xaa281, Xaa326, Xaa386, Xaa388, Xaa390, Xaa405, Xaa426 , Xaa430, Xaa432, Xaa433, Xaa435, Xaa438, Xaa465, Xaa468, Xaa488, Xaa489, Xaa490, Xaa497, Xaa501, Xaa505.
  • the replacement of the amino acid residue at the specified position is selected from the following substitution modes:
  • the substrate can be promoted to enter the activation center to improve the catalytic activity; or the reaction by-products can be reduced and the product purity can be improved.
  • the naturally occurring monooxygenase AcPSMO is capable of oxidizing anisole (a compound of formula IIa) to benzosulfoxide (a compound of formula IIb), but oxidizing omeprazole sulfide (a compound of formula IIIa) is the activity of the compound of formula IIIb Very low.
  • the present invention analyzes the crystal structure of monooxygenase AcPSMO, predicts the entry and exit channels of the substrate, and finds that there is a significant bottleneck part in the entry channel of the compound of formula IIIa, in which residues L143, K326, L426, F432 , T433, S438 and F505 are the key factors affecting the entry of the compound of formula IIIa into the catalytic center.
  • the catalytic substrate specificity of the monooxygenase AcPSMO mutant obtained from the above-mentioned key site modification is changed, and the activity of converting the compound of formula IIIa into the compound of formula IIIb can be increased by more than 5 times.
  • An exemplary monooxygenase AcPSMO mutant amino acid sequence is selected from: SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26; it is relative to the monooxygenase
  • the oxidative activity of AcPSMO (SEQ ID NO: 2) for the compound of formula IIIa is shown in Table 2; the compound of (S)-formula IIIb is omeprazole.
  • the present invention is based on the resolved crystal structure of the monooxygenase AcPSMO, and found that the key sites that affect the selectivity of the substrate and the generation of the compound IIIc by-products are F277, L248, L244, and F281.
  • the single-oxygenase AcPSMO mutants engineered against the above key sites reduce the production of by-product compounds of formula IIIc and increase the ratio of the activity of compounds of formula IIIa to the activity of compounds of formula IIIb by SEQ ID NO: 22 1.5 times or more.
  • An exemplary monooxygenase AcPSMO mutant amino acid sequence is selected from: SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56;
  • the substrate selectivity improvement factor relative to SEQ ID NO: 22 is shown in Table 3; the compound of formula IIIb in Table 3 is the compound of formula IIIb in S configuration.
  • the present invention provides a high-throughput screening method for flat transparent circles.
  • Substrate Formula IIIa compound solution was laid on a flat plate and showed a milky white opaque state.
  • the colony grew 12h, the expression of the polypeptide converted part of the formula IIIa compound into the product formula IIIb compound.
  • the product formula IIIb compound was gradually produced due to its higher solubility than compound IIIa.
  • Transparent circle Directed evolution was used to construct a mutant library, and enzymes with higher activity were screened based on this transparent circle method.
  • the obtained monooxygenase AcPSMO mutant can convert the compound of formula IIIa to the compound of formula IIIb with 1.5 times greater activity than SEQ ID NO: 40.
  • the amino acid sequence of the oxygenase AcPSMO mutant is selected from: SEQ ID NO: 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 90, 92, 94, 96, 98 .
  • amino acid residues other than the designated position can be further processed by any one or more mutation methods of substitution, deletion, and insertion.
  • the above mutation methods include:
  • substitution (deletion, insertion) of amino acid residues at 1 or 2 or 3 or 4 or 5 refers to the substitution (deletion, insertion) of 1 or 2 or 3 amino acid residues Or 4 or 5.
  • the present invention provides a nucleic acid sequence encoding a monooxygenase AcPSMO mutant.
  • the nucleic acid molecule includes, but is not limited to: extracting a naturally occurring nucleic acid molecule encoding a monooxygenase AcPSMO from an organism.
  • a nucleic acid molecule encoding a single oxygenase AcPSMO mutant prepared by genetic engineering operation of a nucleic acid fragment, or a nucleic acid molecule encoding the single oxygenase AcPSMO mutant obtained by an artificial synthesis method.
  • nucleic acid and “nucleic acid molecule” are used interchangeably herein and refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form.
  • the present invention provides a recombinant expression vector comprising the nucleic acid sequence encoding the single oxygenase AcPSMO mutant as described above. Due to the degeneracy of the codon, the nucleic acid sequence encoding the same monooxygenase AcPSMO mutant may not be unique.
  • the recombinant expression vector can be constructed by linking the nucleic acid sequence encoding the monooxygenase AcPSMO mutant as described above to various suitable vectors by conventional methods in the art.
  • the vector may be various conventional vectors in the art, such as commercially available plasmids, cosmids, phage, or viral vectors; further, the vector is preferably a plasmid, and the recombinant expression vector prepared according to conventional technical means in the art is recombinant expression Plasmid, the more preferred plasmid is plasmid pET28a.
  • the present invention provides a recombinant expression transformant, which contains the recombinant expression vector as described above.
  • the recombinant expression transformant can be prepared by transforming the recombinant expression vector of the present invention into a host cell.
  • the host cell may be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated by itself, and the isolated gene of monooxygenase carried by it can be efficiently expressed.
  • E. coli is preferred, and E. coli BL21 (DE3) is preferred.
  • the PCR product containing the single-oxygenase AcPSMO gene (as shown in SEQ ID NO: 1) amplified by PCR is double-digested with restriction enzymes Nde I and Hind III to form complementary sticky ends, and simultaneously
  • the expression vector pET28a is double-digested with restriction enzymes Nde I and Hind III, and the cleaved gene fragment and expression vector are connected by T4 DNA ligase to generate a recombinant expression plasmid pET28a-AcPSMO containing the monooxygenase gene of the invention ,As shown in Figure 1.
  • coli BL21(DE3) a genetically engineered strain expressing recombinant monooxygenase AcPSMO, namely E. coli BL21(DE3) can be obtained )/pET28a-AcPSMO.
  • a recombinant plasmid expressing the above-mentioned monooxygenase AcPSMO mutant is obtained, and then a genetically engineered strain expressing various mutants of the monooxygenase AcPSMO described in the present invention is obtained.
  • the invention discloses a method for preparing the above monooxygenase, cultivating the above-mentioned recombinant expression transformant, and then separating the monooxygenase therefrom.
  • the culture method and culture conditions of the recombinant expression transformant described in the present invention are not particularly limited, and can be appropriately selected according to the common knowledge in the art according to the factors such as the type of host cell and the culture method, as long as the transformant can grow and produce efficiently
  • the single oxygenase AcPSMO mutant of the present invention may be sufficient.
  • the recombinant expression transformant of the present invention is Escherichia coli, it is preferable to culture the recombinant expression transformant and induce enzyme production in LB medium.
  • the medium contains peptone 10g/L, yeast extract 5g/L, and NaCl 10g/L. pH7.0.
  • the recombinant E. coli (preferably E. coli BL21 (DE3)) involved in the present invention is inoculated into kanamycin-containing LB Cultivation in the culture medium, when the optical density of the culture solution OD 600 reaches 0.5 to 0.7 (preferably 0.6), isopropyl- ⁇ -D-sulfur at a final concentration of 0.1 to 1.0 mmol/L (preferably 0.2 mmol/L)
  • the induction of IPTG and 50-100 mg/L vitamin B can efficiently express the recombinant monooxygenase of the present invention.
  • the monooxygenase AcPSMO mutant can be isolated from the recombinant expression transformant by conventional technical means in the art.
  • An exemplary method is to centrifuge the fermentation broth of recombinant E. coli (including but not limited to shake flask culture, fermentor culture) to collect the recombinant E. coli cells.
  • KPB buffer potassium phosphate buffer
  • the supernatant enzyme liquid is taken by centrifugation of the broken liquid, and the supernatant enzyme liquid can also be made into lyophilized enzyme powder by freeze drying.
  • Exemplary ultrasonic crushing conditions are that the set power of the ultrasonic crusher is 400 W, working for 4 s, and intermittent for 6 s, with a total of 99 cycles.
  • Exemplary high-pressure homogenization crushing conditions are 700-
  • the invention also provides an application of the above-mentioned monooxygenase AcPSMO mutant in asymmetric catalytic oxidation of latent chiral sulfide compounds.
  • the monooxygenase catalytically oxidizes the latent chiral sulfide compound into a sulfoxide compound.
  • the latent chiral sulfide compound is selected from compounds of formula IIIa or any of the following chemical formulas:
  • the compounds of the above formulas IIIa, IVa, Va, VIa, and VIIa are respectively omeprazole sulfide, lansoprazole sulfide, pantoprazole sulfide, rabeprazole sulfide, Iprazole sulfide is named, of course, in other literature can also be named in different ways.
  • the asymmetric oxygenation of latent chiral sulfide compounds to sulfoxide compounds using the monooxygenase AcPSMO mutant of the present invention can be The selection of conventional conditions for such reactions in the art. Further, the asymmetric catalytic oxidation reaction can be carried out under shaking or stirring conditions.
  • the harvested supernatant enzyme solution or lyophilized enzyme powder is suspended in a potassium phosphate buffer solution with a pH of 8.5 to 10, a substrate formula IIIa solution is added, and reacted at 20 to 35°C for 6 to 48 hours .
  • a co-solvent is used to solubilize the thioether substrates, such as methanol, ethanol, acetonitrile, isopropanol, acetone, t-butanol, dimethylformamide (DMF), and dimethylsulfoxide (DMSO) , Adding ratio is 2% ⁇ 10% (volume ratio); At the same time, adding hydroxypropyl- ⁇ -cyclodextrin, polyethylene glycol, Triton, Span or Tween additives to make the substrate dispersion better, adding ratio is 0.1% ⁇ 1.5% (weight ratio).
  • a cofactor regeneration system is used to regenerate NADP + to NADPH, for example, formate dehydrogenase is used to oxidize formate to produce CO 2 or alcohol dehydrogenase is used to oxidize isopropanol to produce acetone to achieve the regeneration of the reducing cofactor.
  • the activity of the monooxygenase AcPSMO or its mutants to the compound of formula IIIa or compound of formula IIIb can be determined by the following method: reacting 0.5 mL containing 1 mmol/L of compound of formula IIIa or compound of formula IIIb and 0.2 mmol/L of NADPH The system (100mmol/L KPB buffer, pH 9.0) was preheated to 30°C, then the appropriate amount of thioether monooxygenase was added, and the reaction was incubated at 30°C for shaking and the formation of the product was detected on liquid chromatography.
  • the conversion rate of the monooxygenase AcPSMO or its mutant oxidized formula IIIa compound of the present invention can be determined as follows: 2mmol/L formula IIIa compound and 2mmol/L NADPH 0.5mL reaction system (100mmol/L KPB buffer, pH9. 0, 2% (v/v) DMSO) preheated to 30 °C, then add the appropriate amount of thioether monooxygenase, 30 °C incubation reaction, the formation of the product was detected on liquid chromatography.
  • the present invention adopts protein engineering methods to carry out multiple rounds of molecular transformation of monooxygenase AcPSMO.
  • the constructed monooxygenase AcPSMO mutant can convert the compound of formula IIIa into the compound of formula IIIb, and has higher activity, thermal stability and product Yield.
  • the upstream and downstream primers are designed as follows:
  • the underlined part of the upstream primer is the Nde I restriction site
  • the underlined part of the downstream primer is the Hind III site.
  • PCR amplification was performed using the genomic DNA of Acinetobacter calcoaceticus WP_045432192.1 as a template.
  • the PCR system was: 2 ⁇ Taq PCR MasterMix 25 ⁇ L, upstream primer and downstream primer (10 ng/ ⁇ L) each 2.5 ⁇ L, genomic DNA (100 ng/ ⁇ L) 1 ⁇ L and ddH 2 O 19 ⁇ L.
  • the PCR amplification program is: pre-denaturation at 95°C for 5 minutes, followed by 32 cycles: denaturation at 94°C for 30 seconds, annealing at 50°C for 40 seconds, extension at 72°C for 1.5 minutes; and finally extension at 72°C for 10 minutes.
  • the target fragment is recovered with a DNA recovery kit. After DNA sequencing, the full length of the open reading frame encoded in this sequence is 1629 bp, and its base sequence is shown in SEQ ID NO: 1.
  • the single-oxygenase AcPSMO target fragment and pET28a empty plasmid obtained by PCR amplification in Example 1 were simultaneously digested with restriction enzymes Nde I and Hind III overnight, and then subjected to agarose gel Electrophoresis purification, DNA kit recovery.
  • the recovered target fragment and the empty plasmid vector were ligated at 4°C for 12 hours under the action of T4 DNA ligase to obtain the recombinant plasmid pET28a-AcPSMO.
  • the resulting recombinant plasmid was transformed into E. coli BL21 (DE3), spread on an LB medium plate containing 50 ⁇ g/mL kanamycin, and cultured at 37° C. for 12 to 16 hours. Colony PCR was performed on the grown colonies. Picking colonies and PCR amplifying positive clones with the target band of about 1629bp in length. After sequencing verification, recombinant expression transformant E.coli BL21(DE3)/pET28a-AcPSMO was obtained.
  • the recombinant expression transformant E.coli BL21(DE3)/pET28a-AcPSMO obtained in Example 2 was inoculated into LB medium containing 50 ⁇ g/mL kanamycin, shaken at 37°C for 12 hours, and then shaken 1% (v/v) inoculation volume was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium, placed at 37°C, 180rpm shaker shaking culture, when the OD 600 of the culture solution reached 0.6, add IPTG to the end
  • the concentration was 0.1 mmol/L and the final concentration of complex vitamin B was 70 mg/L. After induction at 16° C.
  • the culture solution was centrifuged at 15000 rpm for 5 minutes.
  • the cells were collected and washed with physiological saline to obtain resting cells.
  • the resulting cells were resuspended in 10 mL of KPB buffer (100 mM, pH 9.0), and subjected to the following ultrasonic treatment in an ice-water bath: 400 W power, working for 4 s, intermittent 6 s, performing 99 cycles, centrifuging at 15000 rpm for 40 min at 4° C, collecting Clear enzyme solution, freeze-dried to obtain freeze-dried enzyme powder.
  • the activity was measured according to the method described in Example 8 (determination of the oxidative activity of the compound of formula IIIa), and the activity was 58 U/g lyophilized enzyme powder.
  • the buffers used in the purification process of Ni affinity self-packing column are: Solution A: 50mM KPB pH8.0, 500mM NaCl, 10mM imidazole, 2mM ⁇ -mercaptoethanol; Solution B: 50mM KPB pH8.0, 500mM NaCl, 300mM imidazole, 2mM ⁇ -mercaptoethanol; Solution C: 50mM KPB pH9.0, 150mM NaCl, 1mM DTT.
  • the purification method is as follows:
  • Ni column is pre-equilibrated with 5-10 times column volume A solution
  • step c) The supernatant stored in step a) is loaded;
  • the collected target protein is concentrated with a 30kDa ultrafiltration tube;
  • step f) The protein obtained in step f) is loaded onto the gel column, the loading volume is 2 mL, and the protein concentration is controlled within 10 mg/mL;
  • step (1) Place the high-purity enzyme solution obtained in step (1) on ice to thaw and centrifuge at 4°C to remove the precipitate.
  • the PreCrystallization Test (PCT) kit was used to screen the protein crystal concentration, and 14 mg/mL was finally selected as the crystal protein concentration according to the protein precipitation. After diluting the target protein to this concentration with solution C, the initial screening of crystals is started.
  • PCT PreCrystallization Test
  • a goniometer of the BL17U or BL19U X-ray diffractometer of the Shanghai Synchrotron Radiation Center (Shanghai Light Source, Shanghai Synchrotron Radiation Facility) with a crystal transfer tool such as cryotong, and adjust the position of the crystal.
  • Collect X-ray diffraction data The collected diffraction data is in the form of a graph, and the data is preprocessed by HKL2000.
  • the preprocessing includes three steps: Index, Integrate and Scale. After these three steps, sca and log format files will be generated, which can be used for the subsequent processing of crystal data.
  • PCR reaction procedure pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for 30 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 7.5 minutes; finally extension at 72°C for 5 minutes.
  • the PCR product was transferred to E.coli BL21 (DE3) competent cells and spread on kanamycin-containing plates, and placed in a 37°C incubator for about 12 static cultures. ⁇ 16h.
  • the plasmid was extracted using the kit. Using this plasmid as a template, SED ID NO: 87 and SED ID NO: 88 as upstream and downstream primers, after repeating the above PCR, digestion and transformation steps, the resulting monoclonal colonies were sequenced, and the base sequence is as SEQ ID NO: As shown in 3, the amino acid sequence of the expressed monooxygenase AcPSMO mutant is shown in SEQ ID NO:4.
  • GGCCCGAACGGTCCG CTG ACCAACCTGCCGCCG
  • amino acid sequence is shown in SEQ ID NO: 6 (the corresponding base sequence is shown in SEQ ID NO: 5);
  • amino acid sequence is shown in SEQ ID NO: 8 (the corresponding base sequence is shown in SEQ ID NO: 7);
  • amino acid sequence is shown in SEQ ID NO: 10 (the corresponding base sequence is shown in SEQ ID NO: 9);
  • amino acid sequence is shown in SEQ ID NO: 12 (the corresponding base sequence is shown in SEQ ID NO: 11);
  • amino acid sequence is shown in SEQ ID NO: 14 (the corresponding base sequence is shown in SEQ ID NO: 13);
  • amino acid sequence is shown in SEQ ID NO: 16 (the corresponding base sequence is shown in SEQ ID NO: 15);
  • amino acid sequence is shown in SEQ ID NO: 18 (the corresponding base sequence is shown in SEQ ID NO: 17);
  • amino acid sequence is shown in SEQ ID NO: 20 (the corresponding base sequence is shown in SEQ ID NO: 19);
  • amino acid sequence is shown in SEQ ID NO: 22 (the corresponding base sequence is shown in SEQ ID NO: 21);
  • amino acid sequence is shown in SEQ ID NO: 24 (the corresponding base sequence is shown in SEQ ID NO: 23);
  • amino acid sequence is shown in SEQ ID NO: 26 (the corresponding base sequence is shown in SEQ ID NO: 25);
  • amino acid sequence is shown in SEQ ID NO: 28 (the corresponding base sequence is shown in SEQ ID NO: 27);
  • amino acid sequence is shown in SEQ ID NO: 30 (the corresponding base sequence is shown in SEQ ID NO: 29);
  • amino acid sequence is shown in SEQ ID NO: 32 (the corresponding base sequence is shown in SEQ ID NO: 31);
  • amino acid sequence is shown in SEQ ID NO: 34 (the corresponding base sequence is shown in SEQ ID NO: 33);
  • amino acid sequence is shown in SEQ ID NO: 36 (the corresponding base sequence is shown in SEQ ID NO: 35);
  • amino acid sequence is shown in SEQ ID NO: 38 (the corresponding base sequence is shown in SEQ ID NO: 37);
  • amino acid sequence is shown in SEQ ID NO: 40 (the corresponding base sequence is shown in SEQ ID NO: 39);
  • amino acid sequence is shown in SEQ ID NO: 42 (the corresponding base sequence is shown in SEQ ID NO: 41);
  • amino acid sequence is shown in SEQ ID NO: 44 (the corresponding base sequence is shown in SEQ ID NO: 43);
  • amino acid sequence is shown in SEQ ID NO: 46 (the corresponding base sequence is shown in SEQ ID NO: 45);
  • amino acid sequence is shown in SEQ ID NO: 48 (the corresponding base sequence is shown in SEQ ID NO: 47);
  • amino acid sequence is shown in SEQ ID NO: 50 (the corresponding base sequence is shown in SEQ ID NO: 49);
  • amino acid sequence is shown in SEQ ID NO: 52 (the corresponding base sequence is shown in SEQ ID NO: 51);
  • amino acid sequence is shown in SEQ ID NO: 54 (the corresponding base sequence is shown in SEQ ID NO: 53);
  • amino acid sequence is shown in SEQ ID NO: 56 (the corresponding base sequence is shown in SEQ ID NO: 55).
  • the solution appears milky white.
  • the substrate is consumed and the product is produced during the reaction, the solution will gradually change to a transparent direction.
  • a high-throughput plate transparent circle screening method that can be used to judge the oxidation activity of the compound of formula IIIa was established.
  • the screening plate was an LB solid plate, and 50 ⁇ g/mL kanamycin, 0.1 mM final concentration IPTG, 2 mM compound of formula IIIa and 1% v/v co-solvent DMSO were added.
  • the screening plate was cultured in a constant temperature incubator at 30°C for more than 12 hours, and the generation of transparent circles was observed. Potentially positive clones with obvious transparent circles (larger than the blank control) were selected. The photo is shown in Figure 5.
  • the PCR reaction conditions are as follows: In a PCR reaction system with a total volume of 50 ⁇ L, a template 0.5 to 20 ng, 5 ⁇ L 10 ⁇ PCR buffer (Mg 2+ Plus), 4 ⁇ L dNTP (2.0 mM each), 5 ⁇ L MnCl 2 (1 mM), a pair Mutant primers 2 ⁇ L (10 ⁇ M) each, 0.25 ⁇ L Taq DNA polymerase, add sterile distilled water to 50 ⁇ L.
  • PCR reaction program (1) Denaturation at 95°C for 3min; (2) Denaturation at 94°C for 10sec; (3) Annealing at 60°C for 30sec; (4) Extension at 72°C for 90sec; Steps (2) to (4) 30 cycles in total, Finally, extend the product at 72°C for 10 minutes and store the product at 4°C.
  • the PCR product was verified by agarose gel electrophoresis analysis and then purified by gel excision.
  • the recovered target gene and pET28a were double digested with restriction enzymes Nde I and Hind III at 37°C for 3 to 5 hours.
  • the double-digested product was verified by agarose gel electrophoresis analysis, and then the gel was purified and recovered.
  • the obtained linearized pET28a plasmid and the target gene fragment were ligated at 16°C with T4 DNA ligase.
  • the ligation product was transferred into E.coli BL21 (DE3) competent cells and plated on kanamycin-containing plates, and placed in a 37°C incubator for about 12 to 16 hours.
  • the transformants on the plate were transferred to a plate containing 1 mM kanamycin, 1% (v/v) DMSO, 0.1 mM IPTG, and 2 mM substrate (compound of Formula IIIa), and incubated at 37°C for 12 to 14 hours.
  • Method 6 was used for activity screening, and the transformants producing transparent circles were expanded in test tubes. Purify and characterize mutants with higher activity, and sequence the corresponding genes.
  • amino acid sequence is shown in SEQ ID NO: 58 (the corresponding base sequence is shown in SEQ ID NO: 57);
  • amino acid sequence is shown in SEQ ID NO: 60 (the corresponding base sequence is shown in SEQ ID NO: 59);
  • amino acid sequence is shown in SEQ ID NO: 62 (the corresponding base sequence is shown in SEQ ID NO: 61);
  • amino acid sequence is shown in SEQ ID NO: 64 (the corresponding base sequence is shown in SEQ ID NO: 63);
  • amino acid sequence is shown in SEQ ID NO: 66 (the corresponding base sequence is shown in SEQ ID NO: 65);
  • amino acid sequence is shown in SEQ ID NO: 68 (the corresponding base sequence is shown in SEQ ID NO: 67);
  • amino acid sequence is shown in SEQ ID NO: 70 (the corresponding base sequence is shown in SEQ ID NO: 69);
  • amino acid sequence is shown in SEQ ID NO: 72 (the corresponding base sequence is shown in SEQ ID NO: 71);
  • amino acid sequence is shown in SEQ ID NO: 74 (the corresponding base sequence is shown in SEQ ID NO: 73);
  • amino acid sequence is shown in SEQ ID NO: 76 (the corresponding base sequence is shown in SEQ ID NO: 75);
  • amino acid sequence is shown in SEQ ID NO: 78 (the corresponding base sequence is shown in SEQ ID NO: 77);
  • amino acid sequence is shown in SEQ ID NO: 80 (the corresponding base sequence is shown in SEQ ID NO: 79);
  • amino acid sequence is shown in SEQ ID NO: 82 (the corresponding base sequence is shown in SEQ ID NO: 81);
  • amino acid sequence is shown in SEQ ID NO: 90 (the corresponding base sequence is shown in SEQ ID NO: 89);
  • amino acid sequence is shown in SEQ ID NO: 92 (the corresponding base sequence is shown in SEQ ID NO: 91);
  • amino acid sequence is shown in SEQ ID NO: 94 (the corresponding base sequence is shown in SEQ ID NO: 93);
  • amino acid sequence is shown in SEQ ID NO: 96 (the corresponding base sequence is shown in SEQ ID NO: 95);
  • amino acid sequence is shown in SEQ ID NO: 98 (the corresponding base sequence is shown in SEQ ID NO: 97).
  • the monooxygenase AcPSMO mutant obtained by random mutation is obtained by sequencing to obtain the amino acid sequence and/or nucleic acid sequence.
  • Those skilled in the art can also use the site-directed mutation described in Example Technology to prepare.
  • Assay method add 390 ⁇ L of 50 mM KPB buffer at pH 9.0 to 500 ⁇ L of reaction system, 50 ⁇ L of 10 mM substrate solution (compound of Formula IIIa, final concentration of 1 mM, DMSO-assisted), 10 ⁇ L of 10 mM NADPH (final concentration of 0.2 mM), a certain amount
  • the enzyme solution was placed at 30°C and reacted at 1000 rpm for 10 minutes.
  • the amount of product (compound of formula IIIb) produced was determined by HPLC and the enzyme activity was calculated.
  • the peak times of Formula IIIa and Formula IIIb are 7.5 min and 5.9 min, respectively.
  • Enzyme activity (U) definition the amount of enzyme required to catalyze the production of 1 ⁇ M of product (compound of formula IIIb) per minute.
  • optical purity is generally expressed by the term "enantiomeric excess” or the symbol “ee”, which refers to the excess of one enantiomer relative to the other in the mixture.
  • ee the excess of one enantiomer relative to the other in the mixture.
  • >99% means that the residual substrate or certain isomer content is below the lower detection limit and cannot be accurately determined.
  • the analysis of the ee value can be achieved by chiral liquid chromatography analysis of the extracted product.
  • Exemplary liquid chromatography conditions are: using a chiral column IA column with n-heptane/ethanol (70:30) as the flow Phase, the flow rate is 1.0mL/min, the detection temperature is 40°C, the detection wavelength is 300nm, and the detection time is 20min.
  • the peak time of (S)-formula IIIb compound and (R)-formula IIIb compound were 11.2 min and 16.8 min, respectively.
  • Assay method Add 430 ⁇ L KPB buffer (50 mM, pH 9.0) to 500 ⁇ L cuvette, 50 ⁇ L 1M sodium formate (dissolved in water, final concentration 100 mM), 10 ⁇ L 10 mM NADP + (dissolved in water, final concentration 0.2 mM), 10 ⁇ L appropriate Concentration of enzyme solution. Detect at 340nm at 30°C, record the change of absorption peak ( ⁇ A) within 1min, and calculate the enzyme activity according to the following formula:
  • Enzyme activity (U) ⁇ A ⁇ V ⁇ dilution factor ⁇ 10 3 / ( ⁇ ⁇ l)
  • Enzyme activity (U) ⁇ A ⁇ V ⁇ dilution factor ⁇ 10 3 / ( ⁇ ⁇ l)
  • Monooxygenase AcPSMO mutant V2 (SEQ ID NO: 70) 0.01 g of lyophilized enzyme powder, formate dehydrogenase 2.5U, and substrate (compound of formula IIIa) were added to 10 mL KPB buffer (100 mM, pH 9.0) ) 0.02 g, 1.5 times equivalent of sodium formate, NADP + 0.2 mM, tert-butanol to a final concentration of 10% (v/v). The reaction was stirred at 28°C, 180 rpm, and 100 ⁇ L of intermittent samples were taken. After sampling, 0.6 mL of ethyl acetate was added for extraction.
  • the extract was dried over anhydrous sodium sulfate, the solvent was removed by volatilization, and then 0.5 mL of isopropanol was added to dissolve.
  • the substrate conversion rate and product ee value were analyzed and determined. The conversion rate was greater than 97 at 12 h. %, the ee value of the product ((S)-compound of formula IIIb) is greater than 99%, and the content of by-product sulfone (compound of formula IIIc) is 0.3%.
  • the extract was dried over anhydrous sodium sulfate, the solvent was removed by volatilization, then 0.5 mL of isopropanol was added for dissolution, and the substrate conversion rate and product ee value were analyzed and determined.
  • the conversion rate was greater than 97 at 16 h. %, the ee value of the product ((S)-compound of formula IIIb) is greater than 99%, and the content of by-product sulfone (compound of formula IIIc) is 0.4%.
  • Monooxygenase AcPSMO mutant V3 (SEQ ID NO: 74) 0.01 g lyophilized enzyme powder, formate dehydrogenase 2.5 U, substrate (compound of formula IIIa) was added to 10 mL KPB buffer (100 mM, pH 9.0) 0.1 g, 1.5 times equivalent of sodium formate, NADP + 0.2 mM, methanol to a final concentration of 5% (v/v). The reaction was stirred at 28°C and 180 rpm. During the reaction, the pH was adjusted to 9.0 with 1M NaOH solution, and 100 ⁇ L of intermittent samples were taken. After sampling, 0.6 mL of ethyl acetate was added for extraction.
  • the extract was dried over anhydrous sodium sulfate, the solvent was removed by volatilization, then 0.5 mL of isopropanol was added for dissolution, and the substrate conversion rate and product ee value were analyzed and determined.
  • the conversion rate was greater than 97 at 16 h. %, the ee value of the product ((S)-compound of formula IIIb) is greater than 99%, and the content of by-product sulfone (compound of formula IIIc) is 0.5%.
  • Monooxygenase AcPSMO mutant V4 (SEQ ID NO: 82) lyophilized enzyme powder 5g and formate dehydrogenase 2.5g were added to 0.5L KPB buffer (50mM, pH 9.0) continuously at a flow rate of 7.5g/L Substrate (compound of formula IIIa) was added in a stream for 16 h to a final concentration of 120 g/L of 1.5 times the substrate equivalent of sodium formate, NADP + 0.3 mM, methanol to a final concentration of 10% (v/v), and additive Tween 2%. At 25°C, oxygen was periodically reacted for 36 hours, and 100 ⁇ L of intermittent samples were taken.
  • the extract was dried over anhydrous sodium sulfate, and the solvent was removed by volatilization. Then, 0.5 mL of isopropyl alcohol was added to dissolve. The substrate conversion rate and product ee value were analyzed and determined. The conversion rate was greater than 97 at 20 h. %, the ee value of the product ((S)-compound of formula IIIb) is greater than 99%, and the content of by-product sulfone (compound of formula IIIc) is 0.9%.
  • the invention analyzes the crystal structure of Acinetobacter thioether monooxygenase, establishes a high-throughput flat-screen transparent circle screening method, and combines rational design and high-throughput screening to significantly improve the catalytic activity of the enzyme and the space-time yield of catalytic reaction.
  • the monooxygenase of the present invention has high catalytic activity, the reaction is added as a small amount of catalyst, and the reaction scale is not limited to the laboratory scale, and can be industrialized, providing new biocatalyst resources for the industrial synthesis of chiral sulfoxide drugs.

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Abstract

提供了一种单加氧酶，其氨基酸序列是由SEQ ID NO:2所示的氨基酸序列通过突变获得，所述单加氧酶可制备手性亚砜类药物，用于胃溃疡治疗药质子泵抑制剂的生产。

Description

一种单加氧酶及其在制备光学纯亚砜中的应用 技术领域
本发明涉及一种非天然硫醚单加氧酶以及酶法催化硫醚类化合物不对称氧化制备亚砜化合物的应用。
技术背景
艾司奥美拉唑，又称(S)-奥美拉唑，化学名为5-甲氧基-2-((S)-((4-甲氧基-3,5-二甲基-2-吡啶基)甲基)亚硫酰基)-1H-苯并咪唑，化学结构如式I所示。艾司奥美拉唑是首个应用于临床的质子泵抑制剂奥美拉唑的(S)-单一构型异构体。该药主要用于治疗十二指肠溃荡、胃溃荡、胃炎以及消化道食管炎。临床上已经证明该药比外消旋体和(R)-奥美拉唑的毒副作用低，且疗效要更好。化学法利用金属催化剂不对称氧化硫醚合成艾司奥美拉唑，但这类方法存在光学纯度受限、过度氧化、副产物多、分离提纯工艺复杂等缺点。
Figure PCTCN2019120798-appb-000001
使用酶法来对硫醚化合物进行不对称氧化，得到单一对映体的手性亚砜，这种方法环境污染小，副产物少，原子经济性好，而且产物光学纯度高，因此利用酶法不对称氧化合成手性亚砜受到了越来越多的关注。
发明人也曾筛选获得一株红球菌(CGMCC2547)可催化一系列潜手性苯烷基硫醚及其衍生物的不对称氧化，获得了光学活性的手性苯甲亚砜及其衍生物(CN101372676B)。捷克科学家筛选获得了一株短小芽孢杆菌Lysinibacillus sp.，利用其生长细胞可催化奥美拉唑硫醚生成(S)-奥美拉唑，但当底物浓度仅为0.1g/L时，44h转化率仅为43％(Bioresources Technology 2011,102:7621–7626)。专利WO2011071982对环己酮单加氧酶NCIMB9871进行了定向进化，该工程酶催化100g/L底物上载量，分5批补加冻干酶粉10g/L，底物在反应69h后实现转化，ee值达到99％。在专利WO2011071982公布的突变序列157基础上，专利CN108118035A公开了将突变体157第386位丝氨酸突变成天冬酰胺，第435位丝氨酸突变成苏氨酸，获得的突变体催化底物浓度高达165g/L。虽然这些可以实现不对称氧化奥美拉唑硫醚合成艾司奥美拉唑，但仍然存在着催化活 力低、催化剂加入量大、反应规模局限于实验室规模等问题。
发明内容
本发明提供的一种单加氧酶，所述单加氧酶包括如SEQ ID NO:2所示的氨基酸序列的突变体，所述突变体的突变方式包括指定位置的氨基酸残基替换，所述指定位置包括如SEQ ID NO:2所示氨基酸序列的Xaa21，Xaa40，Xaa55，Xaa70Xaa143，Xaa145，Xaa156，Xaa185，Xaa220，Xaa244，Xaa246，Xaa248，Xaa249，Xaa277，Xaa281，Xaa326，Xaa386，Xaa388，Xaa390，Xaa405，Xaa426，Xaa430，Xaa432，Xaa433，Xaa435，Xaa438，Xaa465，Xaa468，Xaa488，Xaa489，Xaa490，Xaa497，Xaa501以及Xaa505。
在其中一个事实方式中，所述指定位置的氨基酸残基替换包括至少2处指定位置的氨基酸残基替换。
在其中的一种实施方式中，所述单加氧酶还可选地包括以下任意一种或多种突变方式，所述突变方式包括：
(a)指定位置以外的氨基酸残基替换任意1处或2处或3处或4处或5处；
(b)指定位置以外的氨基酸残基缺失任意1处或2处或3处或4处或5处；
(c)指定位置以外的氨基酸残基插入任意1处或2处或3处或4处或5处。
在其中的一种实施方式中，所述指定位置的氨基酸残基替换包括以下任意一种或几种替换方式：
Xaa21由S替换为G；Xaa40由T替换为A；Xaa55由L替换为Y、W、F或N；Xaa70由E替换为G；Xaa143由L替换为P、或A；Xaa145由A替换为S；Xaa156由E替换为G；Xaa185由G替换为A或S；Xaa220由M替换为R；Xaa244由L替换为V、或I；Xaa246由F替换为Y；Xaa248由L替换为E、N、A、或W；Xaa249由N替换为S；Xaa277由F替换为L、V、Y、I、或D；Xaa281由F替换为V、或A；Xaa326由K替换为C、或F；Xaa386由N替换为S；Xaa388由I替换为F、C、K、G；Xaa390由M替换为S、V、I；Xaa405由K替换为M；Xaa426由L替换为F、或P；Xaa430由G替换为T、或S；Xaa432由F替换为L、或I；Xaa433由T替换为C、或A；Xaa435由L替换为S；Xaa438由S替换为I；Xaa465由K替换为R；Xaa468由V替换为A；Xaa488由E替换为K；Xaa489由S替换为C；Xaa490由W替换为R；Xaa497由P替换为S；Xaa501由N替换为Y；Xaa505由F替换为L。
在其中的一种实施方式中，所述指定位置的氨基酸残基替换包括以下任意一种替换方式：
(1)替换2处，Xaa326由K替换为C，Xaa432由F替换为L；(2)替换2处，Xaa326由 K替换为F，Xaa432由F替换为L；(3)替换2处，Xaa326由K替换为C，Xaa432由F替换为I；
(4)替换4处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa435由L替换为S，Xaa438由S替换为I；
(5)替换4处，Xaa326由K替换为F，Xaa432由F替换为L，Xaa435由L替换为S，Xaa438由S替换为I；
(6)替换5处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa433由T替换为C，Xaa435由L替换为S，Xaa438由S替换为I；
(7)替换5处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
(8)替换6处，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
(9)替换6处，Xaa326由K替换为C，Xaa426由L替换为P，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
(10)替换7处，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(11)替换8处，Xaa143由L替换为P，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(12)替换8处，Xaa143由L替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(13)替换8处，Xaa244由L替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(14)替换8处，Xaa244由L替换为I，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(15)替换9处，Xaa143由L替换为P，Xaa248由L替换为E，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S， Xaa438由S替换为I，Xaa505由F替换为L；
(16)替换8处，Xaa248由L替换为N，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(17)替换8处，Xaa248由L替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(18)替换8处，Xaa248由L替换为W，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(19)替换9处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(20)替换9处，Xaa143由L替换为P，Xaa277由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(21)替换9处，Xaa143由L替换为P，Xaa277由F替换为Y，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(22)替换8处，Xaa277由F替换为I，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(23)替换8处，Xaa277由F替换为D，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(24)替换8处，Xaa281由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(25)替换8处，Xaa281由F替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I， Xaa505由F替换为L；
(26)替换10处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa281由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(27)替换10处，Xaa143由L替换为P，Xaa277由F替换为V，Xaa281由F替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(28)替换11处，Xaa143由L替换为P，Xaa200由M替换为R，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa465由K替换为R，Xaa505由F替换为L；
(29)替换10处，Xaa143由L替换为P，Xaa185由G替换为A，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(30)替换10处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(31)替换11处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa405由K替换为M，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa501由N替换为Y，Xaa505由F替换为L；
(32)替换10处，Xaa143由L替换为P，Xaa156由E替换为G，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(33)替换10处，Xaa143由L替换为P，Xaa249由N替换为S，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(34)替换10处，Xaa40由T替换为A，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
(35)替换11处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S， Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
(36)替换12处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
(37)替换13处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
(38)替换14处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
(39)替换15处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为F，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
(40)替换16处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
(41)替换16处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa488由E替换为K，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
(42)替换19处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432 由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
(43)替换21处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L；
(44)替换22处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa145由A替换为S，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L；
(45)替换23处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa145由A替换为S，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa430由G替换为T，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L。
本发明还提供了一种单加氧酶，所述单加氧酶的氨基酸序列：如SEQ ID NO:4所示，或如SEQ ID NO:6所示，或如SEQ ID NO:8所示，或如SEQ ID NO:10所示，或如SEQ ID NO:12所示，或如SEQ ID NO:14所示，或如SEQ ID NO:16所示，或如SEQ ID NO:18所示，或如SEQ ID NO:20所示，或如SEQ ID NO:22所示，或如SEQ ID NO:24所示，或如SEQ ID NO:26所示，或如SEQ ID NO:28所示，或如SEQ ID NO:30所示，或如SEQ ID NO:32所示，或如SEQ ID NO:34所示，或如SEQ ID NO:36所示，或如SEQ ID NO:38所示，或如SEQ ID NO:40所示，或如SEQ ID NO:42所示，或如SEQ ID NO:44所示，或如SEQ ID NO:46所示，或如SEQ ID NO:48所示，或如SEQ ID NO:50所示，或如SEQ ID NO:52所示，或如SEQ ID NO:54所示，或如SEQ ID NO:56所示，或如SEQ ID NO:58所示，或如SEQ ID NO:60所示，或如SEQ ID NO:62所示，或如SEQ ID NO:64所示，或如SEQ ID NO:66所示，或如SEQ ID NO:68 所示，或如SEQ ID NO:70所示，或如SEQ ID NO:72所示，或如SEQ ID NO:74所示，或如SEQ ID NO:76所示，或如SEQ ID NO:78所示，或如SEQ ID NO:80所示，或如SEQ ID NO:82所示，或如SEQ ID NO:90所示，或如SEQ ID NO:92所示，或如SEQ ID NO:94所示，或如SEQ ID NO:96所示，或如SEQ ID NO:98所示。
本发明还提供了一种分离的核酸，所述核酸编码上述的单加氧酶。
本发明还提供了一种重组表达载体，所述重组表达载体包含如上所述的核酸。
本发明一种重组表达转化体，所述重组表达转化体包含如上所述的重组表达载体。
本发明还提供了一种如上所述的单加氧酶的制备方法，包括以下步骤：培养如上所述的重组表达转化体，从中分离所述单加氧酶。
本发明还提供了一种如上所述的单加氧酶在不对称催化氧化潜手性硫醚化合物为亚砜化合物中的应用。
在其中的一个实施方式中，所述潜手性硫醚化合物选自以下任一化学式所示的化合物：
Figure PCTCN2019120798-appb-000002
需要说明的是本发明中“Xaa数字”表示氨基酸序列中第“数字”位的氨基酸残基，例如 “Xaa40”表示氨基酸序列中第“40”位的氨基酸残基。
本发明中“氨基酸残基(a)+Xaa数字”表示氨基酸序列中第“数字”位的氨基酸残基为“氨基酸残基(a)”，也可简写为“氨基酸残基(a)+数字”；例如“K326”表示氨基酸序列中第“326”位的氨基酸残基为“K”(赖氨酸)。
本发明中“氨基酸残基(a)+Xaa数字+氨基酸残基(b)”表示氨基酸序列中第“数字”位的氨基酸残基由“氨基酸残基(a)”替换为“氨基酸残基(b)”，也可简写为“氨基酸残基(a)+数字+氨基酸残基(b)”；例如“K326C”表示氨基酸序列中第“326”位的氨基酸残基由“K”(赖氨酸)替换为“C”(半胱氨酸)。
本发明中，除非有特别说明，否则“Xaa数字”、“氨基酸残基(a)+Xaa数字”、“氨基酸残基(a)+数字”、“氨基酸残基(a)+Xaa数字+氨基酸残基(b)”、“氨基酸残基(a)+数字+氨基酸残基(b)”中的“数字”，是指以SEQ ID NO:2中的氨基酸位点为基准。
为了克服现有技术的缺陷，本发明解析不动杆菌硫醚单加氧酶的晶体结构，建立高通量的平板透明圈筛选方法，结合理性设计和高通量筛选，获得的单加氧酶催化活力高，反应作为催化剂加入量小，显著提高了酶的催化活力和催化反应的时空产率，反应规模不仅仅局限于实验室规模，为手性亚砜药物的工业合成提供了新的生物催化剂资源。
本发明与现有不对称氧化的其他方法相比，使用本发明的单加氧酶制备手性亚砜类药物具有反应条件温和、环境友好、产率高、产品光学纯度高、过氧化产物少等优势，在胃溃疡治疗药质子泵抑制剂的生产中具有很好的工业应用前景。
附图说明
图1为重组质粒pET28a-AcPSMO的构建图；
图2为单加氧酶AcPSMO的底物通道及其关键位点图；
图3为单加氧酶AcPSMO结构中影响副产物产生的关键位点图；
图4为结晶级纯化SDS-PAGE图，其中泳道1为破碎液，2为Ni洗脱液，3为分子筛上样前样品，4-12为分子筛分离后样品；
图5为平板透明圈筛选法的构建图，其中左图中“+”和“-”分别表示AcPSMO突变体和AcPSMO(SEQ ID NO:2)，右图为实际突变体库的筛选。
具体实施方式
本发明通过生物信息学的方法，分析预测可能对硫醚具有氧化活性的单加氧酶基因，成功 对多个单加氧酶基因进行克隆表达，并验证其功能；其中部分验证结果如表1所示。其中从醋酸钙不动杆菌(Acinetobacter calcoaceticus)中克隆的酶序列WP_045432192.1(NCBI Reference Sequence:WP_045432192.1；https://www.ncbi.nlm.nih.gov/)，重组表达的硫醚单加氧酶可高效催化苯甲硫醚(式IIa化合物)氧化为苯甲亚砜(式IIb化合物)，将该硫醚单加氧酶命名为AcPSMO，其氨基酸序列如SEQ ID NO:2所示。
表1硫醚单加氧酶催化苯甲硫醚为苯甲亚砜
Figure PCTCN2019120798-appb-000003
Figure PCTCN2019120798-appb-000004
本发明采用的醋酸钙不动杆菌(Acinetobacter calcoaceticus)保藏在中国微生物菌种保藏委员会普通微生物中心(China General Microbiological Culture Collection Center，CGMCC，北京市朝阳区北辰西路1号院3号)，保藏时间为：2014年03月20日，其保藏编号为：CGMCC No.8936。该菌株在提交保藏时，保藏证明文件中菌株命名为不动杆菌(Acinetobacter sp.)。Acinetobacter表示属名，calcoaceticus表示种名，sp.表示种名未定，在提交保藏后至本专利申请提交之日，发明人将生物保藏证明中的不动杆菌(Acinetobacter sp.)进一步鉴定到钟，即为醋酸钙不动杆菌(Acinetobacter calcoaceticus)。
天然存在的单加氧酶AcPSMO催化加氧制备某些亚砜化合物的催化活力较低，例如氧化奥美拉唑硫醚(式IIIa化合物)为式IIIb化合物等，本发明通过解析单加氧酶AcPSMO的晶体结构，创造性地发现，某些位点的氨基酸残基是影响底物进入催化中心、影响催化活力的关键，并通过大量的研究，提供了一种单加氧酶，该单加氧酶的氨基酸序列是由SEQ ID NO:2所示的氨基酸序列通过包括替换指定位置的氨基酸残基的任意2处或2处以上的获得；例如可以是通过替换指定位置的氨基酸残基的任意2处、3处、4处、5处、6处、7处、8处、9处、10处、11处、12处、13处、14处、15处、16处、17处、18处、19处、20处、21处、22处、23处、24处、25处、 26处、27处、28处、29处、30处、31处、32处、33处、或34处。
指定位置的氨基酸残基选自：Xaa21，Xaa40，Xaa55，Xaa70，Xaa143，Xaa145，Xaa156，Xaa185，Xaa220，Xaa244，Xaa246，Xaa248，Xaa249，Xaa277，Xaa281，Xaa326，Xaa386，Xaa388，Xaa390，Xaa405，Xaa426，Xaa430，Xaa432，Xaa433，Xaa435，Xaa438，Xaa465，Xaa468，Xaa488，Xaa489，Xaa490，Xaa497，Xaa501，Xaa505。
进一步可选地，替换指定位置的氨基酸残基选自包括以下替换方式：
Xaa21由S替换为G；Xaa40由T替换为A；Xaa55由L替换为Y、W、F或N；Xaa70由E替换为G；Xaa143由L替换为P、或A；Xaa145由A替换为S；Xaa156由E替换为G；Xaa185由G替换为A或S；Xaa220由M替换为R；Xaa244由L替换为V、或I；Xaa246由F替换为Y；Xaa248由L替换为E、N、A、或W；Xaa249由N替换为S；Xaa277由F替换为L、V、Y、I、或D；Xaa281由F替换为V、或A；Xaa326由K替换为C、或F；Xaa386由N替换为S；Xaa388由I替换为F、C、K、G；Xaa390由M替换为S、V、I；Xaa405由K替换为M；Xaa426由L替换为F、或P；Xaa430由G替换为T、或S；Xaa432由F替换为L、或I；Xaa433由T替换为C、或A；Xaa435由L替换为S；Xaa438由S替换为I；Xaa465由K替换为R；Xaa468由V替换为A；Xaa488由E替换为K；Xaa489由S替换为C；Xaa490由W替换为R；Xaa497由P替换为S；Xaa501由N替换为Y；Xaa505由F替换为L。
通过上述替换方式，或可以促进底物进入活化中心，提高催化活力；或可以减少反应副产物，提高产物纯度。
天然存在的单加氧酶AcPSMO能够氧化苯甲硫醚(式IIa化合物)转化为苯甲亚砜(式IIb化合物)，但氧化奥美拉唑硫醚(式IIIa化合物)为式IIIb化合物的活性很低。请参阅图2所示，本发明解析了单加氧酶AcPSMO的晶体结构，预测了底物的进出通道，发现式IIIa化合物进入通道存在明显的瓶颈部分，其中残基L143、K326、L426、F432、T433、S438和F505是影响底物式IIIa化合物进入催化中心的关键。
Figure PCTCN2019120798-appb-000005
Figure PCTCN2019120798-appb-000006
在一些实施方案中，针对上述关键位点改造获得的单加氧酶AcPSMO突变体催化底物特异性发生了改变，能够将式IIIa化合物转化为式IIIb化合物的活力提高5倍以上。示例性的单加氧酶AcPSMO突变体氨基酸序列选自：SEQ ID NO:4、6、8、10、12、14、16、18、20、22、24、26；其相对于单加氧酶AcPSMO(SEQ ID NO:2)对式IIIa化合物的氧化活力如表2所示；(S)-式IIIb化合物即为艾司奥美拉唑。
表2具有改善性质的单加氧酶AcPSMO突变体
Figure PCTCN2019120798-appb-000007
在酶催化氧化式IIIa化合物的反应过程中会过度氧化式IIIb化合物，产生副产物式IIIc化合物，最终导致目标产物式IIIb化合物的纯度和产量的大幅度下降，不利于工业生产。请参阅图3所示，本发明基于解析的单加氧酶AcPSMO晶体结构，发现了影响底物选择性和过氧化副产物式IIIc化合物生成的关键位点是F277、L248、L244和F281。
Figure PCTCN2019120798-appb-000008
在一些实施方案中，针对上述关键位点改造获得的单加氧酶AcPSMO突变体降低副产物式IIIc化合物的产生，对式IIIa化合物活力与式IIIb化合物氧化活力比值相比SEQ ID NO:22提高1.5倍以上。示例性的单加氧酶AcPSMO突变体氨基酸序列选自：SEQ ID NO:28、30、32、34、36、38、40、42、44、46、48、50、52、54、56；其相对于SEQ ID NO:22的底物选择性提高倍数如表3所示；表3中式IIIb化合物为S构型式IIIb化合物。
表3具有降低副产物产生的单加氧酶AcPSMO突变体
Figure PCTCN2019120798-appb-000009
基于底物式IIIa化合物和产物式IIIb化合物的溶解度差异，本发明提供了平板透明圈高通量 筛选方法。底物式IIIa化合物溶液平铺在平板上呈现出乳白色不透明状态，当菌落生长12h以后表达多肽将部分式IIIa化合物转化为产物式IIIb化合物，产物式IIIb化合物因溶解度比化合物IIIa更高，逐渐产生透明圈。采用定向进化构建突变体库，基于此透明圈法筛选活性更高的酶。
在一些实施方案中，基于上述高通量透明圈筛选方法，获得的单加氧酶AcPSMO突变体能够以大于SEQ ID NO:40活力1.5倍将式IIIa化合物转化为式IIIb化合物，所述单加氧酶AcPSMO突变体的氨基酸序列选自：SEQ ID NO:58、60、62、64、66、68、70、72、74、76、78、80、82、90、92、94、96、98。
表4基于高通量筛选获得的具有改善性质的单加氧酶AcPSMO突变体
Figure PCTCN2019120798-appb-000010
Figure PCTCN2019120798-appb-000011
可选地，针对指定位置以外的氨基酸残基，可以通过替换、缺失、插入任意一种或多种突变方式进一步地处理。上述突变方式包括：
(a)指定位置以外的氨基酸残基替换任意1处或2处或3处或4处或5处；
(b)指定位置以外的氨基酸残基缺失任意1处或2处或3处或4处或5处；
(c)指定位置以外的氨基酸残基插入任意1处或2处或3处或4处或5处。
需要说明的是，此处氨基酸残基的替换(缺失、插入)1处或2处或3处或4处或5处是指替换(缺失、插入)氨基酸残基1个或2个或3个或4个或5个。
本发明提供了一种编码单加氧酶AcPSMO突变体的核酸序列，所述核酸分子包括但不限于：从生物体内提取天然存在的编码单加氧酶AcPSMO的核酸分子，通过基因克隆技术对已有核酸片段进行基因工程操作制得的编码单加氧酶AcPSMO突变体的核酸分子，或通过人工合成方法得到的编码所述单加氧酶AcPSMO突变体的核酸分子。术语“核酸”和“核酸分子”在本文中可互换使用，指单链或双链形式的脱氧核糖核苷酸或核糖核苷酸及其聚合物。
本发明提供了一种重组表达载体，该重组表达载体包含编码如上所述的单加氧酶AcPSMO突变体的核酸序列。由于密码子的简并性，编码同一个单加氧酶AcPSMO突变体的核酸序列有可能不是唯一的。所述重组表达载体可通过本领域常规方法将编码如上所述的单加氧酶AcPSMO突变体的核酸序列连接于各种合适载体上构建而成。其中，载体可以是本领域的各种常规载体，如市售的质粒、粘粒、噬菌体或病毒载体等；进一步地，载体优选质粒，根据本领 域常规的技术手段制备的重组表达载体为重组表达质粒，更优选的质粒为质粒pET28a。
本发明提供了一种重组表达转化体，该重组表达转化体包含如上所述的重组表达载体。可通过将本发明的重组表达载体转化至宿主细胞中来制得重组表达转化体。其中，宿主细胞可以是本领域的各种常规宿主细胞，前提是能使该重组表达载体稳定地自行复制，且其所携带的单加氧酶的分离基因可被有效表达。本发明优选大肠杆菌，优选大肠杆菌(E.coli)BL21(DE3)。
将通过PCR扩增所得的包含单加氧酶AcPSMO基因(如SEQ ID NO:1所示)的PCR产物用限制性内切酶Nde I和Hind III双酶切，形成互补的粘性末端，同时将表达载体pET28a用限制性内切酶Nde I和Hind III双酶切，经T4 DNA连接酶连接经过酶切的基因片段和表达载体生成含有本发明的单加氧酶基因的重组表达质粒pET28a-AcPSMO，如图1所示。将所述重组表达质粒pET28a-AcPSMO转化至大肠杆菌(E.coli)BL21(DE3)中，即可获得重组表达单加氧酶AcPSMO的基因工程菌株，即大肠杆菌(E.coli)BL21(DE3)/pET28a-AcPSMO。采用类似的方法，可以很方便地获得表达本发明所述的单加氧酶AcPSMO的各种突变体的基因工程菌株；或者以pET28a-AcPSMO或其突变体为模板，通过定点突变或随机突变的方法，获得表达如上所述的单加氧酶AcPSMO突变体的重组质粒，进而获得表达本发明所述的单加氧酶AcPSMO的各种突变体的基因工程菌株。
本发明公开了一种上述单加氧酶的制备方法，培养上述的重组表达转化体，然后从中分离单加氧酶。
本发明所述重组表达转化体的培养方法和培养条件没有特殊的限制，可以根据宿主细胞类型和培养方法等因素的不同按本领域普通知识进行适当的选择，只要使转化体能够生长并高效产生本发明所述单加氧酶AcPSMO突变体即可。当本发明所述重组表达转化体是大肠杆菌时，优选LB培养基进行重组表达转化体的培养以及诱导产酶，该培养基含有蛋白胨10g/L，酵母膏5g/L，NaCl 10g/L，pH7.0。重组表达转化体的培养和单加氧酶AcPSMO突变体的产生，可优选下述方法：将本发明涉及的重组大肠杆菌(优选E.coli BL21(DE3))接种至含卡那霉素的LB培养基中培养，当培养液的光密度OD 600达到0.5～0.7(优选0.6)时，在终浓度为0.1～1.0mmol/L(优选0.2mmol/L)的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)和50～100mg/L维生素B的诱导下，即可高效表达本发明的重组单加氧酶。
可以通过本领域常规的技术手段从所述重组表达转化体分离获得单加氧酶AcPSMO突变体。示例性的方法为：将重组大肠杆菌的发酵液(包括但不限于摇瓶培养、发酵罐培养)离心，收集重组大肠杆菌细胞。细胞用磷酸钾缓冲液(KPB缓冲液，例如100mM、pH＝9.0)重悬，然后进行超声破碎或高压匀浆破碎。破碎液离心取上清酶液，上清酶液还可以通过冷冻干燥制成 冻干酶粉。示例性的超声破碎条件为，超声破碎仪的设定功率为400W，工作4s，间歇6s，共循环99次。示例性的高压匀浆破碎条件为，700～800bar,2cycles。
本发明还提供了一种上述单加氧酶AcPSMO突变体在不对称催化氧化潜手性硫醚化合物中的应用。优选地，所述单加氧酶将所述潜手性硫醚化合物不对称催化氧化为亚砜化合物。优选地，所述潜手性硫醚化合物选自式IIIa化合物或以下任一化学式所示的化合物：
Figure PCTCN2019120798-appb-000012
其中，本发明中将上述式IIIa、IVa、Va、VIa、VIIa的化合物分别以奥美拉唑硫醚、兰索拉唑硫醚、泮托托拉唑硫醚、雷贝拉唑硫醚、艾普拉唑硫醚进行命名，当然，在其他文献中也可以以不同的命名方式命名。
利用本发明所述单加氧酶AcPSMO突变体将潜手性硫醚化合物不对称催化氧化为亚砜化合物，所涉及的具体反应条件如底物浓度、pH、缓冲液组成、酶用量等可按本领域此类反应的常规条件进行选择。进一步地，不对称催化氧化反应可在振荡或搅拌条件下进行。
在一些实施方案中，将收获的上清酶液或者冻干酶粉，悬浮于pH为8.5～10的磷酸钾缓冲溶液中，加入底物式IIIa溶液，在20～35℃反应6～48小时。
在一些实施方案中，采用共溶剂助溶硫醚底物，例如甲醇、乙醇、乙腈、异丙醇、丙酮、叔丁醇、二甲基甲酰胺(DMF)和二甲基亚砜(DMSO)，添加比例为2％～10％(体积比)；同时添加羟丙基-β-环糊精、聚乙二醇、Triton、Span或吐温添加剂使底物分散更好，添加比例为0.1％～1.5％(重量比)。
在一些实施方案中，采用辅因子再生系统将NADP +再生为NADPH，例如利用甲酸脱氢酶氧化甲酸盐生成CO 2或者醇脱氢酶氧化异丙醇生成丙酮实现还原性辅因子的再生。
本发明所述单加氧酶AcPSMO或其突变体对式IIIa化合物或式IIIb化合物的活力可用如下方法测定：将含1mmol/L式IIIa化合物或式IIIb化合物和0.2mmol/L NADPH的0.5mL反应体系(100mmol/L KPB缓冲液，pH9.0)预热至30℃，然后加入适量的硫醚单加氧酶，30℃保温震荡反应，在液相色谱上检测产物的生成。
本发明所述单加氧酶AcPSMO或其突变体氧化式IIIa化合物转化率可用如下方法测定：2mmol/L式IIIa化合物和2mmol/L NADPH的0.5mL反应体系(100mmol/L KPB缓冲液，pH9.0，2％(v/v)DMSO)预热至30℃，然后加入适量的硫醚单加氧酶，30℃保温震荡反应，在液相色谱上检测产物的生成。
本发明采用蛋白质工程手段对单加氧酶AcPSMO进行多轮分子改造，构建的单加氧酶AcPSMO突变体可以将式IIIa化合物转化为式IIIb化合物，并且具有更高的活性、热稳定性和产物得率。
以下实施例是对本发明进行的进一步的阐述，而不是对本发明保护范围的限制。如无特别说明，所用的原料和试剂均为普通市售产品，所采取的方法和操作方式均为本领域常规方式。
实施例1单加氧酶AcPSMO的基因克隆
根据单加氧酶AcPSMO的开放阅读框，设计上、下游引物如下：
上游引物SEQ ID NO:83：
GGGAATTC CATATGATGACCCAGAAGATGGACTT
下游引物SEQ ID NO:84：
CCC AAGCTTTTAGCTTTCGATCAGGTTGG
其中，上游引物划线部分为Nde I酶切位点，下游引物划线部分为Hind III酶切位点。
以醋酸钙不动杆菌(Acinetobacter calcoaceticus)WP_045432192.1的基因组DNA为模板，进行PCR扩增。PCR体系为：2×Taq PCR MasterMix 25μL，上游引物和下游引物(10ng/μL)各2.5μL，基因组DNA(100ng/μL)1μL和ddH 2O 19μL。PCR扩增程序为：95℃预变性5分钟后进行32次如下循环：94℃变性30秒，50℃退火40秒，72℃延伸1.5分钟；最后72℃再延伸10分钟。PCR扩增产物进行凝胶电泳纯化后，用DNA回收试剂盒回收目的片段。经过DNA测序，该序列中编码的开放阅读框全长1629bp，其碱基序列如SEQ ID NO:1所示。
实施例2单加氧酶AcPSMO重组表达质粒和重组表达转化体的制备
如图1所示，将实施例1中PCR扩增所得的单加氧酶AcPSMO目的片段以及pET28a空质粒同时用限制性内切酶Nde I和Hind III双酶切过夜，然后经琼脂糖凝胶电泳纯化、DNA试 剂盒回收。将回收的经酶切目的片段和空质粒载体在T4 DNA连接酶的作用下，于4℃连接12小时，得到重组质粒pET28a-AcPSMO。
将所得重组质粒转化至E.coli BL21(DE3)，涂布到含有50μg/mL卡那霉素的LB培养基平板上，37℃培养12～16小时，对长出来的菌落进行菌落PCR验证，挑取菌落PCR扩增出长度约1629bp的目的条带的阳性克隆。测序验证后获得重组表达转化体E.coli BL21(DE3)/pET28a-AcPSMO。
实施例3单加氧酶酶AcPSMO的诱导表达及活力测定
将实施例2中所得的重组表达转化体E.coli BL21(DE3)/pET28a-AcPSMO，接种至含50μg/mL卡那霉素的LB培养基中，37℃摇床振荡培养12小时，之后按1％(v/v)的接种量接种至装有100mL的LB培养基的500mL三角瓶中，放入37℃、180rpm摇床振荡培养，当培养液的OD 600达到0.6时，加入IPTG至终浓度0.1mmol/L、复合维生素B终浓度70mg/L进行诱导，16℃诱导24h后，将培养液以15000rpm转速离心5分钟，收集细胞，并用生理盐水洗涤，得到静息细胞。将所得细胞用10mL的KPB缓冲液(100mM,pH9.0)重悬，冰水浴中进行如下超声处理：400W功率，工作4s，间歇6s，进行99个循环，4℃下15000rpm离心40min，收集上清酶液，冻干制得冻干酶粉。按实施例8所述方法进行活力测定(对式IIIa化合物氧化活力的测定)，活力为58U/g冻干酶粉。
实施例4单加氧酶AcPSMO的纯化、结晶和结构解析
(1)结晶级蛋白纯化
Ni亲和自装柱纯化过程中使用的缓冲液为：A液：50mM KPB pH8.0，500mM NaCl，10mM咪唑，2mMβ-巯基乙醇；B液：50mM KPB pH8.0，500mM NaCl，300mM咪唑，2mMβ-巯基乙醇；C液：50mM KPB pH9.0，150mM NaCl，1mM DTT。纯化方法如下：
a)菌体用A液重悬后超声破碎，破碎后的液体用低温高速离心机在4℃离心，12000rpm离心45min，离心后的上清液暂时保存在4℃冰箱或冷库里；
b)Ni柱用5～10倍柱体积的A液预先平衡；
c)步骤a)保存的上清液上样；
d)上样完成后用5～10倍柱体积的A和B混合液(10％B液，体积比)洗去杂蛋白；
e)用1个柱体积的B液洗脱目的蛋白并收集；
f)收集到的目的蛋白用30kDa的超滤管进行浓缩；
g)用1个柱体积纯水冲洗分子筛凝胶柱，然后用1个柱体积C液平衡柱子，流速设为0.5mL/min(可以根据柱压进行调整)；
h)步骤f)获得的蛋白上样至凝胶柱，上样体积2mL，蛋白浓度控制在10mg/mL以内；
i)洗脱：用C液洗脱蛋白，根据280nm处的出峰时间收集目标蛋白，收集到的蛋白通过SDS-PAGE验证纯度，如图4所示；
j)纯度达到要求的样品合并后用30kDa超滤管超滤浓缩至合适浓度，分装后用液氮速冻，并保存于-80℃超低温冰箱。
(2)晶体初筛与条件优化
将步骤(1)得到的高纯度酶液放置于冰上解冻并于4℃离心去除沉淀。在晶体生长条件筛选之前先用Pre Crystallization Test(PCT)试剂盒进行蛋白结晶浓度的筛选，根据蛋白的沉淀情况最终选择14mg/mL作为结晶蛋白浓度。将目标蛋白用C液稀释至该浓度后开始晶体初筛。
首先将结晶试剂盒中的结晶试剂分装到96孔座滴平板中，每个槽中分装75μL；在每个座滴小孔中加入1μL稀释好的酶液和1μL对应槽液，充分混匀但注意不要有气泡产生；用密封膜将加好的结晶初筛板密封后，放入18℃恒温结晶培养箱中；晶体生长一段时间(三天)以后，定期用SX10型显微镜观察晶体生长情况；如果发现初筛板中有晶体长出的条件，记录该条件并查找对应的槽液成分，并在24孔结晶复筛板中进行条件优化，主要是优化晶体生长的pH、沉淀剂浓度、盐浓度等，每个复筛板上优化一个初筛条件；从晶体复筛板上用大小合适的cryoloop捞取生长较好的单颗晶体，快速放置于冷冻保护剂中，在冷冻保护剂中平衡一定时间后迅速放置于液氮中冷冻保存。这里我们对冷冻保护剂进行了优化，分别优化了不同浓度的甘油、PEG和重油，最后选择15％的甘油作为冷冻保护剂。
(3)晶体的X-ray衍射数据采集和处理
将冻存于液氮中的晶体用cryotong等晶体转移工具放置于上海同步辐射中心(上海光源，Shanghai Synchrotron Radiation Facility)BL17U或BL19U X-ray衍射仪的测角器上，并调整晶体的位置后收集X-ray衍射数据。收集到的衍射数据为图谱形式，通过HKL2000进行数据预处理。预处理包括Index、Integrate以及Scale三个步骤，经过这三个步骤处理后会生成sca和log格式文件，可用于晶体数据的后续处理。
后处理中主要用到三个软件：CCP4、Phenix和Coot。具体处理流程如下：
1)在CCP4中用scalepack2mtz程序打开预处理生成的sca文件并将其转换为mtz格式的文件，用Mattews coef程序来计算每个不对称单元中的蛋白分子个数，完成后用Phaser MR程序进行同源分子置换，这里使用RmCHMO(PDB ID:3UCL)为分子置换的模板，最后会生成一个pdb格式文件，这也是目标蛋白的初步三维结构文件；
2)在Phenix软件中对初步得到的结构文件进行自动优化；
3)在Coot软件中通过电子云密度及一级序列中氨基酸残基的骨架对结构进行精修，使R-free、R-work和拉式图等参数达到标准，最后得到单加氧酶AcPSMO的完整晶体结构。
实施例5单加氧酶AcPSMO的定点突变
以pET28a_AcPSMO(实施例2)作为模板，以SED ID NO:85和SED ID NO:86为上下游引物，用Primer Star聚合酶进行高保真PCR。反应体系如下：质粒模板(100ng/μL)，上下游引物(10ng/μL)各0.5μL，DMSO 0.3μL，ddH 2O 8μL，2×Prime Star 10μL。PCR反应程序：95℃预变性3分钟，98℃变性30秒，55℃退火15秒，72℃延伸7.5分钟；最后72℃再延伸5分钟。PCR产物经过Dpn I消化3～5h后将消化产物转入E.coli BL21(DE3)感受态细胞并涂布于含有卡那霉素的平板中，置于37℃培养箱中静置培养约12～16h。将所得到的单克隆菌落进行测序后，利用试剂盒抽取其质粒。以该质粒作为模板，SED ID NO:87和SED ID NO:88为上下游引物重复上述PCR、消化和转化步骤后，将所得到的单克隆菌落进行测序，其碱基序列如SEQ ID NO:3所示，表达的单加氧酶AcPSMO突变体氨基酸序列如SEQ ID NO:4所示。
上游引物SEQ ID NO:85：
ACCGACCTGTACGCG TGCCGTCCGCTGTGCGAT
下游引物SEQ ID NO:86：
ATCGCACAGCGGACG GCACGCGTACAGGTCGGT
上游引物SEQ ID NO:87：
GGCCCGAACGGTCCG CTGACCAACCTGCCGCCG
上游引物SEQ ID NO:88：
CGGCGGCAGGTTGGT CAGCGGACCGTTCGGGCC
采用与获得SEQ ID NO:4所示单加氧酶AcPSMO突变体相似的方法，通过合适的上下游引物，可以获得以下单加氧酶AcPSMO突变体：
氨基酸序列如SEQ ID NO:6所示(相应的碱基序列如SEQ ID NO:5所示)；
氨基酸序列如SEQ ID NO:8所示(相应的碱基序列如SEQ ID NO:7所示)；
氨基酸序列如SEQ ID NO:10所示(相应的碱基序列如SEQ ID NO:9所示)；
氨基酸序列如SEQ ID NO:12所示(相应的碱基序列如SEQ ID NO:11所示)；
氨基酸序列如SEQ ID NO:14所示(相应的碱基序列如SEQ ID NO:13所示)；
氨基酸序列如SEQ ID NO:16所示(相应的碱基序列如SEQ ID NO:15所示)；
氨基酸序列如SEQ ID NO:18所示(相应的碱基序列如SEQ ID NO:17所示)；
氨基酸序列如SEQ ID NO:20所示(相应的碱基序列如SEQ ID NO:19所示)；
氨基酸序列如SEQ ID NO:22所示(相应的碱基序列如SEQ ID NO:21所示)；
氨基酸序列如SEQ ID NO:24所示(相应的碱基序列如SEQ ID NO:23所示)；
氨基酸序列如SEQ ID NO:26所示(相应的碱基序列如SEQ ID NO:25所示)；
氨基酸序列如SEQ ID NO:28所示(相应的碱基序列如SEQ ID NO:27所示)；
氨基酸序列如SEQ ID NO:30所示(相应的碱基序列如SEQ ID NO:29所示)；
氨基酸序列如SEQ ID NO:32所示(相应的碱基序列如SEQ ID NO:31所示)；
氨基酸序列如SEQ ID NO:34所示(相应的碱基序列如SEQ ID NO:33所示)；
氨基酸序列如SEQ ID NO:36所示(相应的碱基序列如SEQ ID NO:35所示)；
氨基酸序列如SEQ ID NO:38所示(相应的碱基序列如SEQ ID NO:37所示)；
氨基酸序列如SEQ ID NO:40所示(相应的碱基序列如SEQ ID NO:39所示)；
氨基酸序列如SEQ ID NO:42所示(相应的碱基序列如SEQ ID NO:41所示)；
氨基酸序列如SEQ ID NO:44所示(相应的碱基序列如SEQ ID NO:43所示)；
氨基酸序列如SEQ ID NO:46所示(相应的碱基序列如SEQ ID NO:45所示)；
氨基酸序列如SEQ ID NO:48所示(相应的碱基序列如SEQ ID NO:47所示)；
氨基酸序列如SEQ ID NO:50所示(相应的碱基序列如SEQ ID NO:49所示)；
氨基酸序列如SEQ ID NO:52所示(相应的碱基序列如SEQ ID NO:51所示)；
氨基酸序列如SEQ ID NO:54所示(相应的碱基序列如SEQ ID NO:53所示)；
氨基酸序列如SEQ ID NO:56所示(相应的碱基序列如SEQ ID NO:55所示)。
实施例6单加氧酶AcPSMO突变体高通量筛选方法的建立
当式IIIa化合物和助溶剂DMSO按一定比例添加时，在溶液中呈现出乳白色，随着反应进行过程中底物消耗及产物产生，溶液会逐渐向透明的方向转变。基于此现象建立了一种可用于判断式IIIa化合物氧化活力的高通量平板透明圈筛选法。筛选平板为LB固体平板，并加入50μg/mL的卡那霉素、0.1mM终浓度的IPTG、2mM式IIIa化合物及1％v/v的助溶剂DMSO。筛选平板在30℃的恒温培养箱中培养12h以上，观察透明圈的产生情况，有明显透明圈(比空白对照大)的潜在阳性克隆子被挑选出来。照片如图5所示。
实施例7硫醚单加氧酶AcPSMO随机突变体的构建
采用易错PCR技术构建硫醚单加氧酶AcPSMO的随机突变库：以实施例5中制备的用于表达SEQ ID NO:40所示单加氧酶AcPSMO突变体的重组质粒作为模板，以For_Nde I和Rev_Hind III为引物，用Taq DNA聚合酶进行易错PCR。为了获得合适的突变率，选择一系列不同的MnCl 2浓度梯度(100μM～300μM的MnCl 2)构建突变库。PCR反应条件如下：总体积为50μL 的PCR反应体系中，加入模板0.5～20ng，5μL 10×PCR buffer(Mg 2+Plus)，4μL dNTP(各2.0mM)，5μL MnCl 2(1mM)，一对突变引物各2μL(10μM)，0.25μL Taq DNA polymerase，加灭菌蒸馏水至50μL。PCR反应程序：(1)95℃变性3min；(2)94℃变性10sec；(3)60℃退火30sec；(4)72℃延伸90sec；步骤(2)～(4)共进行30个循环，最后72℃延伸10min，4℃保存产物。PCR产物经琼脂糖凝胶电泳分析验证后切胶纯化回收，对回收之后的目的基因及pET28a用限制性内切酶Nde I和Hind III在37℃双酶切3～5h。双酶切产物经琼脂糖凝胶电泳分析验证后切胶纯化回收，用T4 DNA连接酶将得到的线性化pET28a质粒与目的基因片段置于16℃进行连接。将连接产物转入E.coli BL21(DE3)感受态细胞并涂布于含有卡那霉素的平板中，置于37℃培养箱中静置培养约12～16h。将平板上的转化子转移至含有1mM卡那霉素、1％(v/v)DMSO、0.1mM IPTG、2mM底物(式IIIa化合物)的平板上，37℃培养12～14h，通过实施例6的方法进行活性筛选，将产生透明圈的转化子进行试管扩大培养。对活性较高的突变体进行纯化表征，对相应基因进行测序。
获得了以下单加氧酶AcPSMO突变体：
氨基酸序列如SEQ ID NO:58所示(相应的碱基序列如SEQ ID NO:57所示)；
氨基酸序列如SEQ ID NO:60所示(相应的碱基序列如SEQ ID NO:59所示)；
氨基酸序列如SEQ ID NO:62所示(相应的碱基序列如SEQ ID NO:61所示)；
氨基酸序列如SEQ ID NO:64所示(相应的碱基序列如SEQ ID NO:63所示)；
氨基酸序列如SEQ ID NO:66所示(相应的碱基序列如SEQ ID NO:65所示)；
氨基酸序列如SEQ ID NO:68所示(相应的碱基序列如SEQ ID NO:67所示)；
氨基酸序列如SEQ ID NO:70所示(相应的碱基序列如SEQ ID NO:69所示)；
氨基酸序列如SEQ ID NO:72所示(相应的碱基序列如SEQ ID NO:71所示)；
氨基酸序列如SEQ ID NO:74所示(相应的碱基序列如SEQ ID NO:73所示)；
氨基酸序列如SEQ ID NO:76所示(相应的碱基序列如SEQ ID NO:75所示)；
氨基酸序列如SEQ ID NO:78所示(相应的碱基序列如SEQ ID NO:77所示)；
氨基酸序列如SEQ ID NO:80所示(相应的碱基序列如SEQ ID NO:79所示)；
氨基酸序列如SEQ ID NO:82所示(相应的碱基序列如SEQ ID NO:81所示)；
氨基酸序列如SEQ ID NO:90所示(相应的碱基序列如SEQ ID NO:89所示)；
氨基酸序列如SEQ ID NO:92所示(相应的碱基序列如SEQ ID NO:91所示)；
氨基酸序列如SEQ ID NO:94所示(相应的碱基序列如SEQ ID NO:93所示)；
氨基酸序列如SEQ ID NO:96所示(相应的碱基序列如SEQ ID NO:95所示)；
氨基酸序列如SEQ ID NO:98所示(相应的碱基序列如SEQ ID NO:97所示)。
需要补充说明的是，本实施例通过随机突变获得的单加氧酶AcPSMO突变体，通过测序获知其氨基酸序列和/或核酸序列后，本领域技术人员也可以通过实施例5所述的定点突变技术来制备获得。
实施例8分析方法
1)单加氧酶AcPSMO及其突变体对式IIIa化合物氧化活力的测定
测定方法：500μL反应体系中加入390μL 50mM、pH 9.0的KPB缓冲液，50μL 10mM底物溶液(式IIIa化合物，终浓度1mM，DMSO助溶)，10μL 10mM NADPH(终浓度0.2mM)，一定量的酶液，置于30℃，1000rpm反应10min。HPLC检测产物(式IIIb化合物)生成的量，计算酶活。示例性的液相色谱条件为：C18反相柱，流动相为乙腈：水＝53:47，流速1mL/min，柱温30℃，检测波长为254nm，检测时间为10min。式IIIa和式IIIb的出峰时间分别是7.5min和5.9min。
酶活(U)定义：每分钟催化生成1μM的产物(式IIIb化合物)所需要的酶量。
2)单加氧酶AcPSMO及其突变体对式IIIb化合物氧化活力的测定
测定方法：500μL反应体系中加入390μL 50mM、pH 9.0的KPB缓冲液，50μL 10mM(S)-式IIIb化合物溶液(终浓度1mM，DMSO助溶)，10μL 10mM NADPH(终浓度0.2mM)，一定量的酶液，置于30℃，1000rpm反应10min。HPLC检测式IIIc化合物生成的量，计算酶活。采用手性柱IA柱，以正庚烷/乙醇(70:30)为流动相，流速为1.0mL/min，检测温度40℃，检测波长为300nm，检测时间为20min。(S)-式IIIb化合物和式IIIc化合物的出峰时间分别是11.2min和8.0min。
酶活(U)定义：每分钟催化生成1μmol的式IIIc化合物所需要的酶量。
3)单加氧酶AcPSMO及其突变体不对称氧化式IIIa化合物的选择性测定
本发明中，光学纯度一般以术语“对映体过量”或符号“ee”表示，该术语是指混合物中一种对映体相对于另一种的过量。除非另有说明，说明书中涉及纯度或ee值时，“>99％”表示残余底物或某异构体含量低于检测下限而无法准确测定。ee值的分析可通过将提取后的产物进行手性液相色谱分析来实现，示例性的液相色谱条件为：采用手性柱IA柱，以正庚烷/乙醇(70:30)为流动相，流速为1.0mL/min，检测温度40℃，检测波长为300nm，检测时间为20min。(S)-式IIIb化合物和(R)-式IIIb化合物的出峰时间分别为11.2min和16.8min。
4)甲酸脱氢酶活力的测定
测定方法：500μL比色皿中加入430μL KPB缓冲液(50mM、pH 9.0)，50μL 1M甲酸钠(溶解于水，终浓度100mM)，10μL 10mM NADP +(溶解于水，终浓度0.2mM)，10μL适当浓度的酶液。在30℃，340nm检测，记录1min内吸收峰的变化值(△A)，根据下列公式计算酶活：
酶活(U)＝△A×V×稀释倍数×10 3/(ε×l)
其中，V是酶液体积，mL；ε(摩尔消光系数)＝6220L·mol -1·cm -1；l为光程，cm。
酶活(U)定义：每分钟生成1μmol NADPH所需要的酶量。
5)异丙醇脱氢酶活力的测定
测定方法：500μL比色皿中加入430μL KPB缓冲液(50mM、pH9.0)，50μL 1M异丙醇(溶解于水，终浓度100mM)，10μL 10mM NADP +(溶解于水，终浓度0.2mM)，10μL适当浓度的酶液。在30℃，340nm，1min内吸收峰的变化值(△A)，根据下列公式计算酶活：
酶活(U)＝△A×V×稀释倍数×10 3/(ε×l)
其中，V是酶液体积，mL；ε(摩尔消光系数)＝6220L·mol- 1·cm- 1；l为光程，cm。
酶活(U)定义：每分钟生成1μmol NADPH所需要的酶量。
实施例9单加氧酶AcPSMO催化氧化苯甲硫醚生成苯甲亚砜
在1mL磷酸钾缓冲液(100mM,pH 9.0)中加入AcPSMO冻干酶粉0.1g(实施例3)，加入2mM化合物IIa、DSMO至终浓度为2％(v/v)，NADP +0.1mM，葡萄糖脱氢酶0.2U，1.5倍底物当量的葡萄糖。在28℃，180rpm搅拌反应，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，进行HPLC检测，24h时转化率大于99％。
实施例10单加氧酶AcPSMO突变体V1催化氧化式IIIa化合物生成艾司奥美拉唑
在100mL KPB缓冲液(100mM,pH8.5)中加入单加氧酶AcPSMO突变体V1(SEQ ID NO:40)冻干酶粉0.1g，葡萄糖脱氢酶15U，加入底物(式IIIa化合物)0.1g，1.5倍当量的葡萄糖，NADP +0.2mM，DMSO至终浓度5％(v/v)。在28℃，180rpm搅拌反应，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，16h时转化率大于99％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量为0.4％。
实施例11单加氧酶AcPSMO突变体V2催化氧化式IIIa化合物生成艾司奥美拉唑
在10mL KPB缓冲液(100mM,pH9.0)中加入单加氧酶AcPSMO突变体V2(SEQ ID NO:70)冻干酶粉0.01g，甲酸脱氢酶2.5U，加入底物(式IIIa化合物)0.02g，1.5倍当量的甲酸钠， NADP +0.2mM，叔丁醇至终浓度10％(v/v)。在28℃，180rpm搅拌反应，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，12h时转化率大于97％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量0.3％。
实施例12单加氧酶AcPSMO突变体V2催化氧化式IIIa化合物生成艾司奥美拉唑
在10mL KPB缓冲液(100mM,pH 9.0)中加入单加氧酶AcPSMO突变体V2(SEQ ID NO:70)冻干酶粉0.03g，葡萄糖脱氢酶2.5U，加入底物(式IIIa化合物)0.05g，1.5倍当量的葡萄糖，NADP +0.2mM，异辛烷至终浓度30％(v/v)。在28℃，180rpm搅拌反应，反应过程以1M NaOH溶液调节pH为9.0，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，16h时转化率大于97％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量0.4％。
实施例13单加氧酶AcPSMO突变体V3催化氧化式IIIa化合物生成艾司奥美拉唑
在10mL KPB缓冲液(100mM,pH 9.0)中加入单加氧酶AcPSMO突变体V3(SEQ ID NO:74)冻干酶粉0.01g，甲酸脱氢酶2.5U，加入底物(式IIIa化合物)0.1g，1.5倍当量的甲酸钠，NADP +0.2mM，甲醇至终浓度5％(v/v)。在28℃，180rpm搅拌反应，反应过程以1M NaOH溶液调节pH为9.0，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，16h时转化率大于97％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量0.5％。
实施例14单加氧酶AcPSMO突变体V4催化氧化式IIIa化合物生成艾司奥美拉唑
在0.5L KPB缓冲液(50mM,pH 9.0)中加入单加氧酶AcPSMO突变体V4(SEQ ID NO:82)冻干酶粉5g，甲酸脱氢酶2.5g，以7.5g/L的流速连续流加入底物(式IIIa化合物)，持续16h，至终浓度120g/L 1.5倍底物当量的甲酸钠，NADP +0.3mM，甲醇至终浓度10％(v/v)，添加剂吐温2％。在25℃，定时通氧气反应36h，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，36h时转化率大于99％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量0.6％。
实施例15单加氧酶AcPSMO突变体V5催化氧化式IIIa化合物生成艾司奥美拉唑
在9L KPB缓冲液(50mM,pH 9.0)中加入单加氧酶AcPSMO突变体V5(SEQ ID NO:98)冻 干酶粉100g，甲酸脱氢酶50g，以7.5g/L的流速连续流加入底物(式IIIa化合物)，持续16h，至终浓度120g/L 1.5倍底物当量的甲酸钠，NADP +0.2mM，甲醇至终浓度10％(v/v)，添加剂吐温2％。在25℃，定时通氧气反应20h，间歇取样100μL。取样后加入0.6mL乙酸乙酯进行萃取，萃取液加入无水硫酸钠干燥，挥发除去溶剂，然后加入0.5mL异丙醇溶解，分析测定底物转化率和产物ee值，20h时转化率大于97％，产物((S)-式IIIb化合物)的ee值大于99％，副产物砜(式IIIc化合物)含量0.9％。
本发明解析不动杆菌硫醚单加氧酶的晶体结构，建立高通量的平板透明圈筛选方法，结合理性设计和高通量筛选显著提高了酶的催化活力和催化反应的时空产率。
本发明的单加氧酶催化活力高，反应作为催化剂加入量小，反应规模不仅仅局限于实验室规模，可以产业化，为手性亚砜药物的工业合成提供了新的生物催化剂资源。

Claims (12)

  1. 一种单加氧酶，其特征在于，所述单加氧酶包括如SEQ ID NO:2所示的氨基酸序列的突变体，所述突变体的突变方式包括指定位置的氨基酸残基替换，所述指定位置选自如SEQ ID NO:2所示氨基酸序列的Xaa21，Xaa40，Xaa55，Xaa70，Xaa143，Xaa145，Xaa156，Xaa185，Xaa220，Xaa244，Xaa246，Xaa248，Xaa249，Xaa277，Xaa281，Xaa326，Xaa386，Xaa388，Xaa390，Xaa405，Xaa426，Xaa430，Xaa432，Xaa433，Xaa435，Xaa438，Xaa465，Xaa468，Xaa488，Xaa489，Xaa490，Xaa497，Xaa501以及Xaa505。
  2. 根据权利要求1所述的单加氧酶，其特征在于，所述指定位置的氨基酸残基替换包括至少2处指定位置的氨基酸残基替换。
  3. 根据权利要求1所述的单加氧酶，其特征在于，所述单加氧酶还包括以下任意一种或多种突变方式，所述突变方式包括：
    (a)指定位置以外的氨基酸残基替换任意1处或2处或3处或4处或5处；
    (b)指定位置以外的氨基酸残基缺失任意1处或2处或3处或4处或5处；
    (c)指定位置以外的氨基酸残基插入任意1处或2处或3处或4处或5处。
  4. 根据权利要求1至3任意一项所述的单加氧酶，其特征在于，所述指定位置的氨基酸残基替换包括以下任意一种或几种替换方式：
    Xaa21由S替换为G；
    Xaa40由T替换为A；
    Xaa55由L替换为Y、W、F或N；
    Xaa70由E替换为G；
    Xaa143由L替换为P、或A；
    Xaa145由A替换为S；
    Xaa156由E替换为G；
    Xaa185由G替换为A或S；
    Xaa220由M替换为R；
    Xaa244由L替换为V、或I；
    Xaa246由F替换为Y；
    Xaa248由L替换为E、N、A、或W；
    Xaa249由N替换为S；
    Xaa277由F替换为L、V、Y、I、或D；
    Xaa281由F替换为V、或A；
    Xaa326由K替换为C、或F；
    Xaa386由N替换为S；
    Xaa388由I替换为F、C、K、G；
    Xaa390由M替换为S、V、I；
    Xaa405由K替换为M；
    Xaa426由L替换为F、或P；
    Xaa430由G替换为T、或S；
    Xaa432由F替换为L、或I；
    Xaa433由T替换为C、或A；
    Xaa435由L替换为S；
    Xaa438由S替换为I；
    Xaa465由K替换为R；
    Xaa468由V替换为A；
    Xaa488由E替换为K；
    Xaa489由S替换为C；
    Xaa490由W替换为R；
    Xaa497由P替换为S；
    Xaa501由N替换为Y；
    Xaa505由F替换为L。
  5. 根据权利要求4所述的单加氧酶，其特征在于，所述指定位置的氨基酸残基替换包括以下任意一种替换方式：
    (1)替换2处，Xaa326由K替换为C，Xaa432由F替换为L；
    (2)替换2处，Xaa326由K替换为F，Xaa432由F替换为L；
    (3)替换2处，Xaa326由K替换为C，Xaa432由F替换为I；
    (4)替换4处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa435由L替换为S，Xaa438由S替换为I；
    (5)替换4处，Xaa326由K替换为F，Xaa432由F替换为L，Xaa435由L替换为S，Xaa438由S替换为I；
    (6)替换5处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa433由T替换为C，Xaa435由L替换为S，Xaa438由S替换为I；
    (7)替换5处，Xaa326由K替换为C，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
    (8)替换6处，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
    (9)替换6处，Xaa326由K替换为C，Xaa426由L替换为P，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I；
    (10)替换7处，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (11)替换8处，Xaa143由L替换为P，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (12)替换8处，Xaa143由L替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (13)替换8处，Xaa244由L替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (14)替换8处，Xaa244由L替换为I，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (15)替换9处，Xaa143由L替换为P，Xaa248由L替换为E，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (16)替换8处，Xaa248由L替换为N，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (17)替换8处，Xaa248由L替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (18)替换8处，Xaa248由L替换为W，Xaa326由K替换为C，Xaa426由L替换为F， Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (19)替换9处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (20)替换9处，Xaa143由L替换为P，Xaa277由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (21)替换9处，Xaa143由L替换为P，Xaa277由F替换为Y，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (22)替换8处，Xaa277由F替换为I，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (23)替换8处，Xaa277由F替换为D，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (24)替换8处，Xaa281由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (25)替换8处，Xaa281由F替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (26)替换10处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa281由F替换为V，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (27)替换10处，Xaa143由L替换为P，Xaa277由F替换为V，Xaa281由F替换为A，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (28)替换11处，Xaa143由L替换为P，Xaa200由M替换为R，Xaa277由F替换为L， Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa465由K替换为R，Xaa505由F替换为L；
    (29)替换10处，Xaa143由L替换为P，Xaa185由G替换为A，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (30)替换10处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (31)替换11处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa405由K替换为M，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa501由N替换为Y，Xaa505由F替换为L；
    (32)替换10处，Xaa143由L替换为P，Xaa156由E替换为G，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (33)替换10处，Xaa143由L替换为P，Xaa249由N替换为S，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (34)替换10处，Xaa40由T替换为A，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa505由F替换为L；
    (35)替换11处，Xaa143由L替换为P，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
    (36)替换12处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
    (37)替换13处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R， Xaa501由N替换为Y，Xaa505由F替换为L；
    (38)替换14处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
    (39)替换15处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为F，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
    (40)替换16处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
    (41)替换16处，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa488由E替换为K，Xaa489由S替换为C，Xaa490由W替换为R，Xaa505由F替换为L；
    (42)替换19处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa501由N替换为Y，Xaa505由F替换为L；
    (43)替换21处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L；
    (44)替换22处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa145由A替换为S，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L；
    (45)替换23处，Xaa21由S替换为G，Xaa55由L替换为Y，Xaa70由E替换为G，Xaa143由L替换为P，Xaa145由A替换为S，Xaa185由G替换为S，Xaa246由F替换为Y，Xaa277由F替换为L，Xaa326由K替换为C，Xaa386由N替换为S，Xaa388由I替换为K，Xaa390由M替换为I，Xaa426由L替换为F，Xaa430由G替换为T，Xaa432由F替换为L，Xaa433由T替换为A，Xaa435由L替换为S，Xaa438由S替换为I，Xaa489由S替换为C，Xaa490由W替换为R，Xaa497由P替换为S，Xaa501由N替换为Y，Xaa505由F替换为L。
  6. 一种单加氧酶，其特征在于，所述单加氧酶的氨基酸序列：
    如SEQ ID NO:4所示，或
    如SEQ ID NO:6所示，或
    如SEQ ID NO:8所示，或
    如SEQ ID NO:10所示，或
    如SEQ ID NO:12所示，或
    如SEQ ID NO:14所示，或
    如SEQ ID NO:16所示，或
    如SEQ ID NO:18所示，或
    如SEQ ID NO:20所示，或
    如SEQ ID NO:22所示，或
    如SEQ ID NO:24所示，或
    如SEQ ID NO:26所示，或
    如SEQ ID NO:28所示，或
    如SEQ ID NO:30所示，或
    如SEQ ID NO:32所示，或
    如SEQ ID NO:34所示，或
    如SEQ ID NO:36所示，或
    如SEQ ID NO:38所示，或
    如SEQ ID NO:40所示，或
    如SEQ ID NO:42所示，或
    如SEQ ID NO:44所示，或
    如SEQ ID NO:46所示，或
    如SEQ ID NO:48所示，或
    如SEQ ID NO:50所示，或
    如SEQ ID NO:52所示，或
    如SEQ ID NO:54所示，或
    如SEQ ID NO:56所示，或
    如SEQ ID NO:58所示，或
    如SEQ ID NO:60所示，或
    如SEQ ID NO:62所示，或
    如SEQ ID NO:64所示，或
    如SEQ ID NO:66所示，或
    如SEQ ID NO:68所示，或
    如SEQ ID NO:70所示，或
    如SEQ ID NO:72所示，或
    如SEQ ID NO:74所示，或
    如SEQ ID NO:76所示，或
    如SEQ ID NO:78所示，或
    如SEQ ID NO:80所示，或
    如SEQ ID NO:82所示，或
    如SEQ ID NO:90所示，或
    如SEQ ID NO:92所示，或
    如SEQ ID NO:94所示，或
    如SEQ ID NO:96所示，或
    如SEQ ID NO:98所示。
  7. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1至6任一项所述的单加氧酶。
  8. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求7所述的核酸。
  9. 一种重组表达转化体，其特征在于，所述重组表达转化体包含如权利要求8所述的重组表达载体。
  10. 一种单加氧酶的制备方法，其特征在于，包括以下步骤：培养如权利要求8所述的重组表达转化体，从中分离所述单加氧酶。
  11. 一种如权利要求1至6任一项所述的单加氧酶在不对称催化氧化潜手性硫醚化合物为亚砜化合物中的应用。
  12. 根据权利要求10所述的应用，其特征在于，所述潜手性硫醚化合物选自以下任一化学式所示的化合物：
    Figure PCTCN2019120798-appb-100001
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