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WO2020211013A1 - 单加氧酶突变体及其应用 - Google Patents

单加氧酶突变体及其应用 Download PDF

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WO2020211013A1
WO2020211013A1 PCT/CN2019/083051 CN2019083051W WO2020211013A1 WO 2020211013 A1 WO2020211013 A1 WO 2020211013A1 CN 2019083051 W CN2019083051 W CN 2019083051W WO 2020211013 A1 WO2020211013 A1 WO 2020211013A1
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Prior art keywords
mutated
pet
group
optionally substituted
alanine
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PCT/CN2019/083051
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English (en)
French (fr)
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洪浩
詹姆斯·盖吉
卢江平
张娜
马玉磊
程逸冰
牟慧艳
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凯莱英生命科学技术(天津)有限公司
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Priority to JP2021551982A priority Critical patent/JP2022523225A/ja
Priority to US17/434,436 priority patent/US20220145341A1/en
Priority to KR1020217037605A priority patent/KR20210153117A/ko
Priority to PCT/CN2019/083051 priority patent/WO2020211013A1/zh
Priority to EP19925142.2A priority patent/EP3957724A4/en
Publication of WO2020211013A1 publication Critical patent/WO2020211013A1/zh
Priority to JP2022170807A priority patent/JP7498244B2/ja

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    • C12Y114/13022—Cyclohexanone monooxygenase (1.14.13.22)

Definitions

  • the invention relates to the field of biotechnology, in particular to a monooxygenase mutant and its application.
  • Cyclohexanone monooxygenase is a reduced coenzyme I (NADPH) dependent oxidase that can catalyze the oxidation of ketones, aldehydes, and some sulfides and selenides.
  • Monooxygenase is widely used in organic synthesis and has good selectivity, controllability and economy. For the synthesis of chiral drugs, usually the product configurations are different, and their functions and toxicity are very different.
  • Thiopenem is a kind of penicillene antibiotics, developed by Pfizer in the United States. It is a penicillin antibiotic for injection. It has a wide antibacterial spectrum, strong antibacterial activity, and is not easily hydrolyzed by ⁇ -lactamase [Antibacterial activity] of sulopenem, a new parenteral penem antibiotic].[J].Japanese Journal of Antibiotics,1996,49(4):338. In the world antibiotic market, it occupies an increasingly important position.
  • the monooxygenase substitution chemical method can be used to carry out the oxidation reaction of (R)-3-hydroxy-tetrahydrothiophene to avoid the chemical exothermic reaction, and at the same time produce high optical purity products (R, R)-1-oxo-3-hydroxytetrahydrothiophene.
  • wild-type monooxygenases have shortcomings such as extremely low selectivity and poor activity, which is far from industrial application.
  • the present invention aims to provide a monooxygenase mutant and its application to solve the technical problems of poor monooxygenase selectivity and low enzyme activity in the prior art.
  • a mutant of monooxygenase is provided.
  • the amino acid sequence of the monooxygenase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO:1, and the mutation includes at least one of the following mutation sites: 49th, 60th, and 61st , 144th, 145th, 146th, 147th, 167th, 169th, 189th, 246th, 247th, 280th, 284th, 285th, 285th 286th, 287th, 328th, 330th, 332th, 382th, 427th, 428th, 429th, 430th, 431th, 432th, 433rd , 434th, 435th, 436th, 438th, 441th, 493rd, 494th, 508th, 509th, 510th, 511th, 512th, and The tryptophan at position 513
  • the mutation includes at least one of the following mutation site combinations: F435A+F508Y; F435S+F508Y; L147Y+F508M; F280Y+F508M; F280Y+F508N; F435A+L510V; F435S+L510V; F435N+L510V; T436A+L510V; L438A+L510V; T436A+L438A; F435A+T436A; F435S+T436A; F435N+T436A; L146Y+F508M; L146F+F508M; F280Y+F508Y; F280Y+F508N; F435A+T436A+F508Y; F435A+T436A+F508M; T436A+L510V; F435S+T436A+L510V; T436A+L438A+F508Y; T436A+
  • a DNA molecule encodes any of the aforementioned monooxygenase mutants.
  • a recombinant plasmid contains any of the aforementioned DNA molecules.
  • the recombinant plasmids are pET-22b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET- 14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a (+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a( +), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+) ), pET-
  • a host cell contains any of the aforementioned recombinant plasmids.
  • the host cells include prokaryotic cells, yeast or eukaryotic cells; preferably, the prokaryotic cells are E. coli BL21 cells or E. coli DH5 ⁇ competent cells.
  • R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; R 1 and R 2 can be singly or combined with each other to form a substituted or unsubstituted ring;
  • R 1 and R 2 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group , More preferably an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group;
  • the aryl group includes phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, and thienyl.
  • the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxy Carbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
  • the aralkyl group is benzyl
  • substitution means substitution by halogen atom, nitrogen atom, sulfur atom, hydroxyl group, nitro group, cyano group, methoxy group, ethoxy group, carboxyl group, carboxymethyl group, carboxyethyl group or methylenedioxy group.
  • R 3 and R 4 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group; R 3 and R 4 can be singly or combined with each other to form a substituted or unsubstituted ring;
  • R 3 and R 4 are an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group , More preferably an optionally substituted or unsubstituted alkyl group having 1-10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group;
  • the aryl group includes phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, and thienyl.
  • the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxy Carbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
  • the aralkyl group is benzyl
  • substitution means substitution by halogen atom, nitrogen atom, sulfur atom, hydroxyl group, nitro group, cyano group, methoxy group, ethoxy group, carboxyl group, carboxymethyl group, carboxyethyl group or methylenedioxy group.
  • the monooxygenase is a solution, lyophilized powder, immobilized enzyme or immobilized cell of any of the above-mentioned monooxygenase mutants.
  • reaction system of the monooxygenation reaction also includes cofactors
  • cofactors are NAD + /NADH and/or NADP + /NADPH
  • cofactor circulation system includes glucose and glucose dehydrogenase, formate and formate dehydrogenase. Hydrogenase, glucose 6-phosphate and glucose-6-phosphate dehydrogenase, or secondary alcohol and secondary alcohol dehydrogenase.
  • the temperature of the monooxygenation reaction is 10 to 37°C, preferably 15 to 35°C.
  • the time for the monooxygenation reaction is 3 to 48 hours, more preferably 6 to 16 hours.
  • the monooxygenation reaction is carried out under the condition of pH 5.0 to 10.0, preferably pH 6.0 to 9.0.
  • the monooxygenase mutant of the present invention is based on the monooxygenase shown in SEQ ID NO: 1, and is mutated through site-directed mutagenesis, thereby changing its amino acid sequence and realizing changes in protein structure and function.
  • the method of directed screening obtains the monooxygenase with the above-mentioned mutation site.
  • the monooxygenase mutant of the present invention has the advantage of greatly improved stereoselectivity, and the enzyme activity is also improved accordingly.
  • the monooxygenase from Rhodococcus sp. can catalyze the reaction of thioether compounds and ketone compounds, but its activity is low and the stereoselectivity is poor, especially for the reaction of (R)-3-hydroxy-tetrahydrothiophene Oxidation, the product obtained is S type, the configuration is opposite to the target product.
  • the present invention strives to improve the stereoselectivity of monooxygenase and the activity of monooxygenase through protein engineering methods, and obtain mutants with improved enzyme catalytic properties. In the production and preparation process of chiral compounds, the usage of enzymes is reduced, Obtain high optical purity products.
  • the inventors of the present invention improved the activity and stereoselectivity of the monooxygenase SEQ ID NO:1 through directed evolution, and reduced the amount of enzyme used. Firstly, a mutation site was introduced into the monooxygenase SEQ ID NO:1 by whole-plasmid PCR, and the mutants were detected for activity and stereoselectivity, and mutants with increased activity or stereoselectivity were selected.
  • SEQ ID NO: 1 as follows: MTAQISPTVVDAVVIGAGFGGIYAVHKLHNEQGLTVVGFDKADGPGGTWYWNRYPGALSDTESHLYRFSFDRDLLQDGTWKTTYITQPEILEYLESVVDRFDLRRHFRFGTEVTSAIYLEDENLWEVSTDKGEVYRAKYVVNAVGLLSAINFPDLPGLDTFEGETIHTAAWPEGKNLAGKRVGVIGTGSTGQQVITALAPEVEHLTVFVRTPQYSVPVGNRPVTKEQIDAIKADYDGIWDSVKKSAVAFGFEESTLPAMSVSEEERNRIFQEAWDHGGGFRFMFGTFGDIATDEAANEAAASFIRSKIAEIIEDPETARKLMPTGLYAKRPLCDNGYYEVYNRPNVEAVAIKENPIREVTAKGVVTEDGVLHELDVLVFATGFDAVDGNYRRIEIRGRNGLHINDHWDGQPTSYLGVTTANFPNWFMVLGPNGPF
  • SEQ ID NO: 1 Using SEQ ID NO: 1 as a template, 60 pairs of site-directed mutation primers were designed (the mutation sites are 49th, 60th, 61st, 144th, 145th, 146th, 147th, 167th, 169th, 189th, 246th, 247th, 280th, 284th, 285th, 286th, 287th, 328th, 330th, 332th , 382nd, 427th, 428th, 429th, 430th, 431th, 432th, 433rd, 434th, 435th, 436th, 438th, and 441th, 493rd, 494th, 508th, 509th, 510th, 511th, 512th, 513th, etc.) using site-directed mutagenesis, expressed as pET-22b(+) Vector to obtain a mutant plasmid with the target gene.
  • the mutation sites are 49th, 60th, 61s
  • site-directed mutagenesis refers to the introduction of desired changes (usually changes in a favorable direction), including bases, into the target DNA fragment (either genome or plasmid) through methods such as polymerase chain reaction (PCR) Add, delete, point mutation, etc.
  • PCR polymerase chain reaction
  • Site-directed mutagenesis can quickly and efficiently improve the traits and characterization of the target protein expressed by DNA, which is a very useful method in gene research.
  • the method of introducing site-directed mutations using whole-plasmid PCR is simple and effective, and is currently a more commonly used method.
  • the principle is that a pair of primers (forward and reverse) containing mutation sites are annealed with the template plasmid and then "circularly extended" with polymerase.
  • the so-called cyclic extension means that the polymerase extends the primers according to the template, and then returns after one turn. Stop at the 5'end of the primer, and then go through a cycle of repeated heating and annealing extension. This reaction is different from rolling circle amplification and will not form multiple tandem copies.
  • the extension products of the forward and reverse primers are annealed and paired to become a nicked open-circle plasmid
  • the template DNA derived from the dam+ strain has methylation sites and can be recognized and cut by Dpn I enzyme, while the plasmid with mutant sequence synthesized in vitro is not digested because it is not methylated.
  • the nick can be repaired naturally, and a clone with a mutant plasmid can be obtained.
  • double-point mutations in combinatorial mutations are the same as that for single-point mutations, using the whole plasmid PCR method. Simultaneously mutate two or more sites of multiple-point mutations by using overlap extension PCR amplification to obtain a mutated gene containing multiple-point mutations. The two ends are digested with restriction enzymes and then connected to the expression vector for transformation Into the Escherichia coli cells, spread on an LB culture dish containing 100 ⁇ g/mL ampicillin, and cultivate overnight at 37° C. to obtain combined mutants, which were identified by sequencing.
  • Overlapping extension PCR technology (gene splicing by overlap extension PCR, referred to as SOEPCR) uses primers with complementary ends to make PCR products form overlapping strands, so that in the subsequent amplification reaction, the overlapping strands extend The augmented fragments are overlapped and spliced together.
  • SOEPCR overlap extension PCR
  • This technology uses PCR technology to perform effective gene recombination in vitro, and is often used in the construction of multiple point mutations.
  • Saturation mutation is a method to obtain mutants in which the target amino acid is replaced by 19 other amino acids in a short time by modifying the coding gene of the target protein. This method is not only a powerful tool for directed modification of proteins, but also an important method for the study of protein structure-function relationships. Saturation mutations can often obtain more ideal evolutionary bodies than single point mutations. For these problems that the site-directed mutation method cannot solve, it is precisely the unique point that the saturation mutation method is good at.
  • a mutant of monooxygenase is provided.
  • the amino acid sequence of the monooxygenase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO:1, and the mutation includes at least one of the following mutation positions: 49th, 60th, and 61st, 144th, 145th, 146th, 147th, 167th, 169th, 189th, 246th, 247th, 280th, 284th, 285th , 286th, 287th, 328th, 330th, 332th, 382th, 427th, 428th, 429th, 430th, 431th, 432th, and 433rd, 434th, 435th, 436th, 438th, 441th, 493rd, 494th, 508th, 509th, 510th, 511th, 512th And the 513th position, and the 49th tryptophan was mutated to
  • mutations include at least one of the following mutation site combinations: F435A+F508Y; F435S+F508Y; L147Y+F508M; F280Y+F508M; F280Y+F508N; F435A+L510V; F435S+L510V; F435N+L510V; T436A+L510V; L438A+L510V; T436A+L438A; F435A+T436A; F435S+T436A; F435N+T436A; L146Y+F508M; L146F+F508M; F280Y+F508Y; F280Y+F508N; FF435A+T436A+508; F435A+T436A+F508M; F435A+T436A+L510V; F435A+T436A+L510V; F435A+T436A+L510V; F4
  • the mutation of phenylalanine at position 435 to alanine and the mutation of phenylalanine at position 508 to tyrosine are described as: F435A+F508Y, and other descriptions are the same.
  • the monooxygenase mutant of the present invention is based on the monooxygenase shown in SEQ ID NO: 1, and is mutated through site-directed mutagenesis, thereby changing its amino acid sequence and realizing changes in protein structure and function.
  • the method of directed screening obtains the monooxygenase with the above-mentioned mutation site.
  • the monooxygenase mutant of the present invention has the advantage of greatly improved enzyme stereoselectivity, and the enzyme activity is also improved accordingly.
  • a DNA molecule is provided.
  • This DNA molecule encodes the aforementioned monooxygenase mutant.
  • the monooxygenase obtained by the above-mentioned DNA encoding improves the enzyme activity and the stereoselectivity of the enzyme, and can reduce the amount of added enzyme in the monooxygenation reaction of thioether compounds or ketone compounds and reduce the difficulty of post-treatment.
  • DNA molecules of the present invention may also exist in the form of "expression cassettes".
  • "Expression cassette” refers to a linear or circular nucleic acid molecule, covering DNA and RNA sequences that can direct the expression of a specific nucleotide sequence in an appropriate host cell. Generally speaking, it includes a promoter operatively linked to the target nucleotide, which is optionally operatively linked to a termination signal and/or other regulatory elements.
  • the expression cassette may also include sequences required for proper translation of the nucleotide sequence.
  • the coding region usually encodes the target protein, but also encodes the target functional RNA in the sense or antisense orientation, such as antisense RNA or untranslated RNA.
  • the expression cassette containing the target polynucleotide sequence may be chimeric, meaning that at least one of its components is heterologous to at least one of the other components.
  • the expression cassette may also be naturally occurring, but obtained by efficient recombination for heterologous expression.
  • a recombinant plasmid is provided.
  • the recombinant plasmid contains any of the aforementioned DNA molecules.
  • the DNA molecule in the recombinant plasmid is placed in an appropriate position of the recombinant plasmid, so that the DNA molecule can be replicated, transcribed or expressed correctly and smoothly.
  • plasmid used in the present invention includes any plasmid, cosmid, bacteriophage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, and may be a prokaryotic expression plasmid or It can be a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid.
  • the recombinant plasmid is selected from pET-22b(+), pET-22b(+), pET-3a(+), pET-3d(+ ), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+) , PET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET -38b(+), pET-39b(+), pET-40b(+),
  • a host cell contains any of the aforementioned recombinant plasmids.
  • Host cells suitable for use in the present invention include but are not limited to prokaryotic cells, yeast or eukaryotic cells.
  • the prokaryotic cells are eubacteria, such as gram-negative bacteria or gram-positive bacteria. More preferably, the prokaryotic cells are E. coli BL21 cells or E. coli DH5 ⁇ competent cells. The best conditions for inducing expression of monooxygenase: 25°C, 0.2mM IPTG induction for 16h.
  • the mutant plasmid was transformed into E. coli cells, and then the crude enzyme was obtained by ultrasonically disrupting the cells.
  • an application of a monooxygenase mutant in catalyzing the monooxygenation reaction of thioether compounds or ketone compounds is provided.
  • the monooxygenase is any of the above-mentioned mutants of monooxygenase. Since the above-mentioned monooxygenase mutant of the present invention has higher enzymatic catalytic activity, the use of the monooxygenase mutant of the present invention for industrial production can not only reduce production costs, but also obtain products with purer chirality.
  • the thioether compound is In the above formula, R 1 and R 2 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group. In addition, R 1 and R 2 may be alone or in combination with each other to form a substituted or unsubstituted ring.
  • R 1 and R 2 are preferably an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group, more Preferred are an optionally substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
  • aryl groups include phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, and thienyl. Oxy, oxadiazolyloxy, imidazolyloxy, thiazolyloxy, furyloxy, pyrrolyloxy and the like.
  • alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxy Alkylcarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl, cycloheptyl, etc.
  • aralkyl group a benzyl group etc. are mentioned. These groups can be further substituted.
  • R 3 and R 4 each independently represent an optionally substituted or unsubstituted alkyl group, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
  • R 3 and R 4 may be alone or in combination with each other to form a substituted or unsubstituted ring.
  • R 3 and R 4 are preferably an optionally substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group, more Preferred are an optionally substituted or unsubstituted alkyl group having 1 to 10 carbon atoms, an optionally substituted or unsubstituted aralkyl group, or an optionally substituted or unsubstituted aryl group.
  • aryl groups include phenyl, naphthyl, pyridyl, thienyl, oxadiazolyl, imidazolyl, thiazolyl, furyl, pyrrolyl, phenoxy, naphthyloxy, pyridyloxy, and thienyl. Oxy, oxadiazolyloxy, imidazolyloxy, thiazolyloxy, furyloxy, pyrrolyloxy and the like.
  • alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, methoxy Alkylcarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl, cycloheptyl, etc.
  • aralkyl group a benzyl group etc. are mentioned. These groups can be further substituted.
  • substituents examples include halogen atoms, nitrogen atoms, sulfur atoms, hydroxyl groups, nitro groups, cyano groups, methoxy groups, ethoxy groups, carboxyl groups, carboxymethyl groups, and carboxyethyl groups. Or methylenedioxy and so on.
  • the formation of the ring may also be via these substituents.
  • the monooxygenase mutant of the present invention is applied to the side chain synthesis of thiopenem, and the reaction formula is as follows:
  • the monooxygenase mutant obtained by the present invention can be used for the synthesis of the side chain of thiopenem, avoiding the exothermic reaction, and obtaining (R, R)-1-oxo-3-hydroxytetrahydrothiophene with high optical purity (Conversion rate>99%, ee value 95.0%), the industrial production cost of the compound is greatly reduced, and the enzyme has better application value in industrial production.
  • the monooxygenase can be a solution of a monooxygenase mutant, a lyophilized powder, an immobilized enzyme, or an immobilized cell.
  • the cofactor that catalyzes the monooxygenation reaction is NAD + /NADH and/or NADP + /NADPH
  • the cofactor circulation system includes glucose and glucose dehydrogenase, formate and formate dehydrogenase, and glucose 6 -Phosphoric acid and glucose-6-phosphate dehydrogenase, secondary alcohol (e.g. isopropanol) and/or secondary alcohol dehydrogenase and similar systems, most preferably isopropanol and alcohol dehydrogenase.
  • the temperature for catalyzing the monooxygenation reaction is 10 ⁇ 37°C, more preferably 15 ⁇ 35°C; the time for catalyzing the monooxygenation reaction is 3 ⁇ 48h, more preferably 6 ⁇ 16h; the catalytic monooxygenation reaction is at pH It is carried out under the condition of 6.0 to 10.0, and more preferably the pH is 6.0 to 9.0; under this reaction condition, the catalytic performance of the enzyme can be better exerted.
  • the conversion rate is 10%-50%+, 50%-90%++, 90%++++; ee value is -99%-50%*, -50%-0% **.
  • Conversion rate is 10%-50%+, 50%-90%++, over 90%+++; ee value is -99% ⁇ -50%*, ee value is -50% ⁇ 0 %**, in the range of 0%-20%***, in the range of 20%-60%****, in the range of 60%-80%*****, in the range of 80%-99%** ****.
  • the activity with a conversion rate between 10% and 50% is +, between 50% and 90% is ++, and when the conversion rate is above 90%, it is +++; if the ee value is between -99% and -50%, it is at -50% ⁇ 0%**, at 0% ⁇ 20%***, at 20% ⁇ 60%****, at 60% ⁇ 80%*****, at 80% ⁇ 99% ******.
  • mutant active e.e. Wild type + * F508Y+F435A+L438A+W49A+D60L +++ **** F508Y+F435A+L438A+V144E +++ **** F508Y+F435A+L438A+G145F +++ **** F508Y+F435A+L438A+T436A+H167W +++ **** F508Y+F435A+L438A+T436A+A169K +++ **** F508Y+F435A+L438A+T436A+S179M +++ **** F508Y+F435A+L438A+T436A+A246V +++ **** F508Y+F435A+L438A+T436A+V247T +++ **** F508Y+F435A+L438A+T436A+V247T +++ **** F508Y+F435
  • the activity with a conversion rate between 10% and 50% is +, between 50% and 90% is ++, and when the conversion rate is above 90%, it is +++; if the ee value is between -99% and -50%, it is at -50% ⁇ 0%**, at 0% ⁇ 20%***, at 20% ⁇ 60%****, at 60% ⁇ 80%*****, at 80% ⁇ 99% ******.
  • reaction results are as follows:
  • the activity with a conversion rate between 10% and 50% is +, between 50% and 90% is ++, and when the conversion rate is above 90%, it is +++; if the ee value is between -99% and -50%, it is at -50% ⁇ 0%**, at 0% ⁇ 20%***, at 20% ⁇ 60%****, at 60% ⁇ 80%*****, at 80% ⁇ 99% ******; If the yield is between 0% and 20%, it is ##, where the yield is between 20% and 40% is ##, and the yield is between 40% and 60% is ###.
  • the activity with a conversion rate between 10% and 50% is +, between 50% and 90% is ++, and when the conversion rate is above 90%, it is +++; if the ee value is between 0% and 20%, it is between 20% and 60%.
  • the ** is between 60% and 80% of ***, and between 80% and 99% of ****.

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Abstract

公开了一种单加氧酶突变体及其应用。该单加氧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生突变得到的氨基酸序列，突变至少包括如下突变位点之一：第49位、第60位、第61位、第144位、第145位、第146位、第147位、第167位、第169位、第189位、第246位、第247位、第280位、第284位、第285位、第286位、第287位、第328位、第330位、第332位、第382位、第427位、第428位、第429位、第430位、第431位、第432位等，该单加氧酶突变体具有立体选择性大幅度提高的优势，并且酶活也有相应提高。

Description

单加氧酶突变体及其应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种单加氧酶突变体及其应用。
背景技术
传统的化学氧化剂大多是有毒和/或易爆的，且立体选择性很低，出于环境友好以及合成高光学纯度药物和农用化学品的需要，人们正不断开发新型氧化剂。环己酮单加氧酶(CHMO)是一种还原型辅酶I(NADPH)依赖型氧化酶，可催化酮、醛以及一些硫化物、硒化物的氧化反应。单加氧酶在有机合成中应用较广，具有较好的选择性、可控性和经济性。对于手性药物的合成，通常产品构型不同，其功能、毒性相差很大，因此，得到高光学纯度的手性化合物在制药研发中非常重要。近几十年来，单加氧酶一直作为生物催化剂被用于催化立体选择性的反应中，进而合成一系列有价值的手性化合物[Protein Engineering of Stereoselective Baeyer—Villiger Monooxygenases].[J].Chemistry-A European Journal,2012,18(33)。
硫培南作为青霉烯类抗生素的一种，由美国Pfizer公司研发，为注射用青霉烯类抗生素，具有抗菌谱广，抗菌活性强，不易被β-内酰胺酶水解等特性[Antibacterial activity of sulopenem,a new parenteral penem antibiotic].[J].Japanese Journal of Antibiotics,1996,49(4):338。在世界抗生素市场中，占有越来越重要的地位。
利用生物酶法进行不对称合成，由于自身高选择性以及环境友好性等优势，越来越受到人们的关注。(R)-3-羟基-四氢噻吩是生产抗生素硫培南等多种药物的关键中间体，在酶催化合成硫培南侧链的方法中(见下图)，我们通过蛋白质改造，已经获得了选择性和活性都大幅提高的酮还原酶突变体，可催化第一步3-酮基四氢噻吩转化为(R)-3-羟基-四氢噻吩[专利：酮还原酶突变体及其应用(公开号CN108048417A)]。在第二步酶催化反应中，可采用单加氧酶取代化学法进行(R)-3-羟基-四氢噻吩的氧化反应，避免化学放热反应，同时生成高光学纯度的产品(R，R)-1-氧-3-羟基四氢噻吩。
Figure PCTCN2019083051-appb-000001
然而对于目前来说，野生型的单加氧酶存在选择性极低，活性较差等缺点，距离工业化应用有较大的差距。一般而言，我们可以通过蛋白质工程的手段对野生酶进行改造，提高酶的立体选择性和活性，从而可以应用在工业生产中。
发明内容
本发明旨在提供一种单加氧酶突变体及其应用，以解决现有技术中单加氧酶选择性差、酶活性低的技术问题。
为了实现上述目的，根据本发明的一个方面，提供了一种单加氧酶突变体。该单加氧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生突变得到的氨基酸序列，突变至少包括如下突变位点之一：第49位、第60位、第61位、第144位、第145位、第146位、第147位、第167位、第169位、第189位、第246位、第247位、第280位、第284位、第285位、第286位、第287位、第328位、第330位、第332位、第382位、第427位、第428位、第429位、第430位、第431位、第432位、第433位、第434位、第435位、第436位、第438位、第441位、第493位、第494位、第508位、第509位、第510位、第511位、第512位和第513位，且突变为第49位的色氨酸突变为丙氨酸；第60位的天冬氨酸突变为亮氨酸；第61位的苏氨酸突变为谷氨酰胺；第144位的缬氨酸突变为谷氨酸；第145位的甘氨酸突变为苯丙氨酸；第146位的亮氨酸突变为苯丙氨酸或酪氨酸；第147位的亮氨酸突变为甲硫氨酸、苏氨酸或酪氨酸；第167位的组氨酸突变为色氨酸；第169位的丙氨酸突变为赖氨酸；第189位的丝氨酸突变为蛋氨酸；第246位的丙氨酸突变为缬氨酸；第247位的缬突变为苏氨酸；第280位的苯丙氨酸突变为酪氨酸、色氨酸或缬氨酸；第284位的苯丙氨酸突变为丝氨酸；第285位的甘氨酸突变为丙氨酸；第286位的苏氨酸突变为丙氨酸；第287位的苯丙氨酸突变为天冬氨酸；第328位的丙氨酸突变为天冬酰胺；第330位的精氨酸突变为丙氨酸；第332位的亮氨酸突变为精氨酸；第382位的甘氨酸突变为丙氨酸；第427位的蛋氨酸突变为异亮氨酸；第428位的缬氨酸突变为丙氨酸；第429位的亮氨酸突变为酪氨酸；第430位的甘氨酸突变为丙氨酸；第431位的脯氨酸突变为丙氨酸；第432位的天冬酰胺突变为酪氨酸；第433位的甘氨酸突变为酪氨酸；第434位的脯氨酸突变为丙氨酸；第435位的苯丙氨酸突变为丝氨酸、丙氨酸、天冬酰胺或酪氨酸；第436位的苏氨酸突变为丙氨酸、丝氨酸、甘氨酸、谷氨酸或半胱氨酸；第438位的亮氨酸突变为甘氨酸、丙氨酸、酪氨酸、苯丙氨酸或丝氨酸；第441位的丝氨酸突变为亮氨酸和缬氨酸；第493位的色氨酸突变为丙氨酸；第494位的异亮氨酸突变为丙氨酸、甲硫氨酸或丝氨酸，第508位的苯丙氨酸突变为酪氨酸、甲硫氨酸或天冬酰胺，第509位的酪氨酸突变为蛋氨酸；第510位的亮氨酸突变为缬氨酸；第511位的甘氨酸突变为亮氨酸；第512位的甘氨酸突变为异亮氨酸；第513位的亮氨酸突变为缬氨酸；或者单加氧酶突变体的氨基酸序列具有发生突变的氨基酸序列中的突变位点，且与发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。
进一步地，突变至少包括如下突变位点组合之一：F435A+F508Y；F435S+F508Y；L147Y+F508M；F280Y+F508M；F280Y+F508N；F435A+L510V；F435S+L510V；F435N+L510V；T436A+L510V；L438A+L510V；T436A+L438A；F435A+T436A；F435S+T436A；F435N+T436A；L146Y+F508M；L146F+F508M；F280Y+F508Y；F280Y+F508N；F435A+T436A+F508Y；F435A+T436A+F508M；F435A+T436A+L510V；F435S+T436A+L510V；T436A+L438A+F508Y；T436A+L438A+F508M；T436A+L438A+F508N；L147Y+F435A+F508M； L147Y+F435A+F508Y；L147Y+F435A+F508N；L147Y+F435S+F508Y；L147Y+F435N+F508Y；L147Y+F435S+F508Y；L147Y+F435N+F508Y；F508Y+F435A+L438A；F508Y+F435A+L438Y；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A+T436A+F280W；F508Y+F435A+L438A+T436A+F280A；F508Y+F435A+L438A+T436A+S441L；F508M+F435A+L438A+T436A+F280W；F508M+F435A+L438A+T436A+F280V；F508M+F435A+L438A+T436A+S441V；F508M+F435A+L438A+T436A+S441A；F508Y+F435N+L438A+T436S；F508Y+F435N+L438A+T436S+F280V；F508Y+F435N+L438A+T436S+S441L；F508Y+F435N+L438A+T436S+F280V+S441V；F508Y+F435N+L438A+T436S+F280V+S441V+L510V；F508M+F435N+L438A+T436S+F280V+S441V+L510V；F508M+F435A+L438A+T436S+F280V+S441V+L510V；F508M+F435S+L438A+T436S+F280V+S441V+L510V；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V；F508Y+F435N+L438A+T436A+F280V+S441V+L510V；F508Y+F435N+L438A+T436A+F280V+S441A+L510V；F508Y+F435N+L438A+T436A+F280V+S441L+L510V；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M；F508Y+F435N+L438A+T436S+F280V+S441V+L510V+D60L；F508M+F435N+L438A+T436S+F280V+S441V+L510V+D60L+T61Q；F508M+F435A+L438A+T436S+F280V+S441V+L510V+D60L+G145F；F508M+F435S+L438A+T436S+F280V+S441V+L510V+D60L+A169K；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L+S189M；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+L332R；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L+A328N；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+G430A；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A+D60L+N432Y；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S+D60L+Y509M；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M+D60L+G512I；F508Y+F435A+L438A+W49A+D60L；F508Y+F435A+L438A+V144E；F508Y+F435A+L438A+G145F；F508Y+F435A+L438A+T436A+H167W；F508Y+F435A+L438A+T436A+A169K；F508Y+F435A+L438A+T436A+S179M；F508Y+F435A+L438A+T436A+A246V；F508Y+F435A+L438A+T436A+V247T；F508Y+F435A+L438A+T436A+F284S；F508Y+F435A+L438A+T436A+G285A； F508M+F435A+L438A+T436A+T286A；F508M+F435A+L438A+T436A+R330A；F508M+F435A+L438A+T436A+L332R；F508M+F435A+L438A+T436A+A328N；F508Y+F435A+L438A+T436S+G382A；F508Y+F435A+L438A+T436S+M427I；F508Y+F435A+L438A+T436S+V428A；F508Y+F435A+L438A+T436S+G430A；F508Y+F435A+L438A+T436S+P431A；F508Y+F435A+L438A+T436S+N432Y；F508Y+F435A+L438A+T436S+G433Y；F508Y+F435A+L438A+T436S+P434A；F508Y+F435A+L438A+T436S+Y509M；F508Y+F435A+L438A+T436S+G511L；F508Y+F435A+L438A+T436S+G512I；F508Y+F435A+L438A+T436S+L513V；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+P431A；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147Y+I494S；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147T+I494M；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A；F508Y+F435A+L438Y；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A+T436A+F280W+D60L；F508Y+F435A+L438A+T436A+F280A+V247T；F508Y+F435A+L438A+T436A+S441L+F285A；F508Y+F435N+L438A+T436S+R330A；F508Y+F435N+L438A+T436S+F280V+G430A；F508Y+F435N+L438A+T436S+S441L+P434A；F508M+F435N+L438A+T436S+F280V+S441V+L510V+Q60L+T286A+Y509M；F508M+F435A+L438A+T436S+F280V+S441V+L510V+Q60L+T61Q+V247T；F508M+F435S+L438A+T436S+F280V+S441V+L510V+Q60L+F287D+R330A；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+V144E+G145F+M427I；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+Q60L+L322R+N432Y；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+R330A+P321A+G512I；和F508Y+F435N+L438A+T436A+F280V+S441L+L510V+T61Q+R330A+G430A。
根据本发明的另一方面，提供了一种DNA分子。该DNA分子编码上述任一种单加氧酶突变体。
根据本发明的再一方面，提供了一种重组质粒。该重组质粒含有上述任一种DNA分子。
进一步地，重组质粒为pET-22b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、 pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。
根据本发明的另一方面，提供了一种宿主细胞。该宿主细胞含有上述任一种重组质粒。
进一步地，宿主细胞包括原核细胞、酵母或真核细胞；优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。
根据本发明的另一方面，提供了一种上述任一种单加氧酶突变体在催化硫醚类化合物或酮类化合物的单加氧反应中的应用。
进一步地，硫醚类化合物为
Figure PCTCN2019083051-appb-000002
其中，R 1和R 2分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；R 1和R 2可单独或两者互相结合形成取代或未被取代的环；
优选的，R 1和R 2为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；
优选的，芳基包括苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基和吡咯基氧基；
优选的，烷基包括甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基和环庚基；
优选的，芳烷基为苄基；
优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
进一步地，酮类化合物为
Figure PCTCN2019083051-appb-000003
其中，R 3和R 4分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；R 3和R 4可单独或两者互相结合形成取代或未被取代的环；
优选的，R 3和R 4为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的 烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；
优选的，芳基包括苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基和吡咯基氧基；
优选的，烷基包括甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基和环庚基；
优选的，芳烷基为苄基；
优选的，取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
进一步地，应用为硫培南侧链合成。
进一步地，单加氧酶为上述任一种单加氧酶突变体的溶液、冻干粉、固定化酶或固定化细胞。
进一步地，单加氧反应的反应体系中还包括辅因子，辅因子为NAD +/NADH和/或NADP +/NADPH，辅因子循环系统包括葡萄糖和葡糖脱氢酶、甲酸盐和甲酸脱氢酶、葡糖6-磷酸和葡糖-6-磷酸脱氢酶，或仲醇和仲醇脱氢酶。
进一步地，单加氧反应的温度为10～37℃，优选为15～35℃。
进一步地，单加氧反应的时间为3～48小时，更优选为6～16小时。
进一步地，单加氧反应在pH为5.0～10.0的条件下进行，优选pH为6.0～9.0。
本发明的单加氧酶突变体是在SEQ ID NO：1所示的单加氧酶基础上，通过定点突变的方法进行突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的单加氧酶，本发明的单加氧酶突变体具有立体选择性大幅度提高的优势，并且酶活也有相应提高。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
来源于Rhodococcus sp.的单加氧酶可以催化硫醚类化合物以及酮类化合物反应，但是其活性较低，立体选择性较差，特别是催化反应(R)-3-羟基-四氢噻吩的氧化，获得的产品为S型,构型与目标产物相反。本发明力图通过蛋白质工程的方法提高单加氧酶的立体选择性，提高单加氧酶的活性，获得酶催化特性改善的突变体，在手性化合物生产制备过程中，降低酶 的使用量，获得高光学纯度的产品。
本发明的发明人通过定向进化的方法提高单加氧酶SEQ ID NO：1的活性与立体选择性性，降低酶的使用量。首先通过全质粒PCR的方式在单加氧酶SEQ ID NO：1上引入突变位点，对突变体进行活性和立体选择性检测，挑选活性或立体选择性提高的突变体。
SEQ ID NO：1如下所示：MTAQISPTVVDAVVIGAGFGGIYAVHKLHNEQGLTVVGFDKADGPGGTWYWNRYPGALSDTESHLYRFSFDRDLLQDGTWKTTYITQPEILEYLESVVDRFDLRRHFRFGTEVTSAIYLEDENLWEVSTDKGEVYRAKYVVNAVGLLSAINFPDLPGLDTFEGETIHTAAWPEGKNLAGKRVGVIGTGSTGQQVITALAPEVEHLTVFVRTPQYSVPVGNRPVTKEQIDAIKADYDGIWDSVKKSAVAFGFEESTLPAMSVSEEERNRIFQEAWDHGGGFRFMFGTFGDIATDEAANEAAASFIRSKIAEIIEDPETARKLMPTGLYAKRPLCDNGYYEVYNRPNVEAVAIKENPIREVTAKGVVTEDGVLHELDVLVFATGFDAVDGNYRRIEIRGRNGLHINDHWDGQPTSYLGVTTANFPNWFMVLGPNGPFTNLPPSIETQVEWISDTVAYAERNEIRAIEPTPEAEEEWTQTCTDIANATLFTRGDSWIFGANVPGKKPSVLFYLGGLGNYRNVLAGVVADSYRGFELKSAVPVTA。以SEQ ID NO：1为模版，设计了60对定点突变引物(突变位点为第49位、第60位、第61位、第144位、第145位、第146位、第147位、第167位、第169位、第189位、第246位、第247位、第280位、第284位、第285位、第286位、第287位、第328位、第330位、第332位、第382位、第427位、第428位、第429位、第430位、第431位、第432位、第433位、第434位、第435位、第436位、第438位、第441位、第493位、第494位、第508位、第509位、第510位、第511位、第512位、第513位等)利用定点突变手段，以pET-22b(+)为表达载体，获得带有目的基因的突变质粒。
其中，定点突变：是指通过聚合酶链式反应(PCR)等方法向目的DNA片段(可以是基因组，也可以是质粒)中引入所需变化(通常是表征有利方向的变化)，包括碱基的添加、删除、点突变等。定点突变能迅速、高效的提高DNA所表达的目的蛋白的性状及表征，是基因研究工作中一种非常有用的手段。
利用全质粒PCR引入定点突变的方法简单有效，是目前使用比较多的手段。其原理是，一对包含突变位点的引物(正、反向)，和模板质粒退火后用聚合酶“循环延伸”，(所谓的循环延伸是指聚合酶按照模版延伸引物，一圈后回到引物5’端终止，再经过反复加热退火延伸的循环，这个反应区别于滚环扩增，不会形成多个串联拷贝。正反向引物的延伸产物退火后配对成为带缺刻的开环质粒。来源于dam+菌株的模版DNA由于带有甲基化位点，可被Dpn I酶识别切割，而体外合成的带突变序列的质粒由于没有甲基化而不被消化，在转化入大肠杆菌后缺刻可被自然修复，即可得到带有突变质粒的克隆。将突变质粒转化至大肠杆菌感受态细胞内，涂布于含有LB固体培养基(100μg/mL氨苄青霉素)的培养皿中，37℃过夜培养。对固体培养基上长出的单克隆进行活化。测序鉴定正确后，在25℃，0.2mM IPTG的条件下，过夜诱导单加氧酶的表达。然后通过离心、超声破碎细胞的方法获得粗酶液，用于反应特性检测。
在单点突变获得催化特性提高的突变体基础上，可对有益位点进行组合，以获得性状更 优的突变体。组合突变中双点突变的构建方法和单点突变的构建方法一样，采用全质粒PCR法构建。同时突变2个及以上位点的多点突变通过采用重叠延伸PCR扩增进行，获得含多点突变的突变基因，两端经限制性内切酶双酶切后，连接到表达载体上，转化至大肠杆菌细胞内，涂布于含有100μg/mL氨苄青霉素的LB培养皿中，37℃培养过夜，获得组合突变体，测序鉴定。
重叠延伸PCR技术(gene splicing by overlap extension PCR，简称SOEPCR)是采用具有互补末端的引物，使PCR产物形成了重叠链，从而在随后的扩增反应中通过重叠链的延伸，将不同来源的扩增片段重叠拼接起来。此技术利用PCR技术能够在体外进行有效的基因重组，常常被用于多点突变的构建中。
通过采用计算机对单加氧酶和底物的三维结构进行对接模拟分析，在酶催化中心附近发现一些氨基酸距离底物为
Figure PCTCN2019083051-appb-000004
可能与酶对底物的立体选择性以及活性有很大关系。可以在多点组合突变的基础上，对这些可能有影响的氨基酸位点进行迭代饱和突变，以获得活性和立体选择性都大幅度提高的突变体。
饱和突变是通过对目的蛋白的编码基因进行改造，短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体的一种方法。此方法不仅是蛋白质定向改造的强有力工具，而且是蛋白质结构－功能关系研究的重要手段。饱和突变往往能获得比单点突变更为理想的进化体。而对于定点突变方法不能解决的这些问题，恰恰是饱和突变方法所擅长的独特之处。
根据本发明一种典型的实施方式，提供一种单加氧酶突变体。该单加氧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生突变得到的氨基酸序列，所述突变至少包括如下突变位点之一：第49位、第60位、第61位、第144位、第145位、第146位、第147位、第167位、第169位、第189位、第246位、第247位、第280位、第284位、第285位、第286位、第287位、第328位、第330位、第332位、第382位、第427位、第428位、第429位、第430位、第431位、第432位、第433位、第434位、第435位、第436位、第438位、第441位、第493位、第494位、第508位、第509位、第510位、第511位、第512位和第513位，且所述突变为第49位的色氨酸突变为丙氨酸；第60位的天冬氨酸突变为亮氨酸；第61位的苏氨酸突变为谷氨酰胺；第144位的缬氨酸突变为谷氨酸；第145位的甘氨酸突变为苯丙氨酸；第146位的亮氨酸突变为苯丙氨酸或酪氨酸；第147位的亮氨酸突变为甲硫氨酸、苏氨酸或酪氨酸；第167位的组氨酸突变为色氨酸；第169位的丙氨酸突变为赖氨酸；第189位的丝氨酸突变为蛋氨酸；第246位的丙氨酸突变为缬氨酸；第247位的缬突变为苏氨酸；第280位的苯丙氨酸突变为酪氨酸、色氨酸或缬氨酸；第284位的苯丙氨酸突变为丝氨酸；第285位的甘氨酸突变为丙氨酸；第286位的苏氨酸突变为丙氨酸；第287位的苯丙氨酸突变为天冬氨酸；第328位的丙氨酸突变为天冬酰胺；第330位的精氨酸突变为丙氨酸；第332位的亮氨酸突变为精氨酸；第382位的甘氨酸突变为丙氨酸；第427位的蛋氨酸突变为异亮氨酸；第428位的缬氨酸突变为丙氨酸；第429位的亮氨酸突变为酪氨酸；第430位的甘氨酸突变为丙氨酸；第431位的脯氨酸突变为丙氨酸；第432位的天冬 酰胺突变为酪氨酸；第433位的甘氨酸突变为酪氨酸；第434位的脯氨酸突变为丙氨酸；第435位的苯丙氨酸突变为丝氨酸、丙氨酸、天冬酰胺或酪氨酸；第436位的苏氨酸突变为丙氨酸、丝氨酸、甘氨酸、谷氨酸或半胱氨酸；第438位的亮氨酸突变为甘氨酸、丙氨酸、酪氨酸、苯丙氨酸或丝氨酸；第441位的丝氨酸突变为亮氨酸和缬氨酸；第493位的色氨酸突变为丙氨酸；第494位的异亮氨酸突变为丙氨酸、甲硫氨酸或丝氨酸，第508位的苯丙氨酸突变为酪氨酸、甲硫氨酸或天冬酰胺，第509位的酪氨酸突变为蛋氨酸；第510位的亮氨酸突变为缬氨酸；第511位的甘氨酸突变为亮氨酸；第512位的甘氨酸突变为异亮氨酸；第513位的亮氨酸突变为缬氨酸；或者所述单加氧酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与所述发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。
经反应验活，获得了ee值和活性提高的突变体。具体而言，优选的，包括如下组合：突变至少包括如下突变位点组合之一：F435A+F508Y；F435S+F508Y；L147Y+F508M；F280Y+F508M；F280Y+F508N；F435A+L510V；F435S+L510V；F435N+L510V；T436A+L510V；L438A+L510V；T436A+L438A；F435A+T436A；F435S+T436A；F435N+T436A；L146Y+F508M；L146F+F508M；F280Y+F508Y；F280Y+F508N；F435A+T436A+F508Y；F435A+T436A+F508M；F435A+T436A+L510V；F435S+T436A+L510V；T436A+L438A+F508Y；T436A+L438A+F508M；T436A+L438A+F508N；L147Y+F435A+F508M；L147Y+F435A+F508Y；L147Y+F435A+F508N；L147Y+F435S+F508Y；L147Y+F435N+F508Y；L147Y+F435S+F508Y；L147Y+F435N+F508Y；F508Y+F435A+L438A；F508Y+F435A+L438Y；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A+T436A+F280W；F508Y+F435A+L438A+T436A+F280A；F508Y+F435A+L438A+T436A+S441L；F508M+F435A+L438A+T436A+F280W；F508M+F435A+L438A+T436A+F280V；F508M+F435A+L438A+T436A+S441V；F508M+F435A+L438A+T436A+S441A；F508Y+F435N+L438A+T436S；F508Y+F435N+L438A+T436S+F280V；F508Y+F435N+L438A+T436S+S441L；F508Y+F435N+L438A+T436S+F280V+S441V；F508Y+F435N+L438A+T436S+F280V+S441V+L510V；F508M+F435N+L438A+T436S+F280V+S441V+L510V；F508M+F435A+L438A+T436S+F280V+S441V+L510V；F508M+F435S+L438A+T436S+F280V+S441V+L510V；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V；F508Y+F435N+L438A+T436A+F280V+S441V+L510V；F508Y+F435N+L438A+T436A+F280V+S441A+L510V；F508Y+F435N+L438A+T436A+F280V+S441L+L510V；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M； F508Y+F435N+L438A+T436S+F280V+S441V+L510V+D60L；F508M+F435N+L438A+T436S+F280V+S441V+L510V+D60L+T61Q；F508M+F435A+L438A+T436S+F280V+S441V+L510V+D60L+G145F；F508M+F435S+L438A+T436S+F280V+S441V+L510V+D60L+A169K；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L+S189M；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+L332R；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L+A328N；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+G430A；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A+D60L+N432Y；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S+D60L+Y509M；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M+D60L+G512I；F508Y+F435A+L438A+W49A+D60L；F508Y+F435A+L438A+V144E；F508Y+F435A+L438A+G145F；F508Y+F435A+L438A+T436A+H167W；F508Y+F435A+L438A+T436A+A169K；F508Y+F435A+L438A+T436A+S179M；F508Y+F435A+L438A+T436A+A246V；F508Y+F435A+L438A+T436A+V247T；F508Y+F435A+L438A+T436A+F284S；F508Y+F435A+L438A+T436A+G285A；F508M+F435A+L438A+T436A+T286A；F508M+F435A+L438A+T436A+R330A；F508M+F435A+L438A+T436A+L332R；F508M+F435A+L438A+T436A+A328N；F508Y+F435A+L438A+T436S+G382A；F508Y+F435A+L438A+T436S+M427I；F508Y+F435A+L438A+T436S+V428A；F508Y+F435A+L438A+T436S+G430A；F508Y+F435A+L438A+T436S+P431A；F508Y+F435A+L438A+T436S+N432Y；F508Y+F435A+L438A+T436S+G433Y；F508Y+F435A+L438A+T436S+P434A；F508Y+F435A+L438A+T436S+Y509M；F508Y+F435A+L438A+T436S+G511L；F508Y+F435A+L438A+T436S+G512I；F508Y+F435A+L438A+T436S+L513V；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+P431A；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147Y+I494S；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147T+I494M；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A；F508Y+F435A+L438Y；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；F508Y+F435A+L438A+T436A+F280W+D60L；F508Y+F435A+L438A+T436A+F280A+V247T；F508Y+F435A+L438A+T436A+S441L+F285A；F508Y+F435N+L438A+T436S+R330A； F508Y+F435N+L438A+T436S+F280V+G430A；F508Y+F435N+L438A+T436S+S441L+P434A；F508M+F435N+L438A+T436S+F280V+S441V+L510V+Q60L+T286A+Y509M；F508M+F435A+L438A+T436S+F280V+S441V+L510V+Q60L+T61Q+V247T；F508M+F435S+L438A+T436S+F280V+S441V+L510V+Q60L+F287D+R330A；F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+V144E+G145F+M427I；F508Y+F435N+L438A+T436A+F280V+S441V+L510V+Q60L+L322R+N432Y；F508Y+F435N+L438A+T436A+F280V+S441A+L510V+R330A+P321A+G512I；和F508Y+F435N+L438A+T436A+F280V+S441L+L510V+T61Q+R330A+G430A；和F508Y+F435N+L438A+T436A+F280V+S441L+L510V+T61Q+R330A+G430A。其中，为了描述方便第435位的苯丙氨酸突变为丙氨酸和第508位的苯丙氨酸突变为酪氨酸描述为：F435A+F508Y，其他描述同理。
本发明的单加氧酶突变体是在SEQ ID NO：1所示的单加氧酶基础上，通过定点突变的方法进行突变，从而改变其氨基酸序列，实现蛋白质结构和功能的改变，再通过定向筛选的方法，得到具有上述突变位点的单加氧酶，本发明的单加氧酶突变体具有酶立体选择性大幅度提高的优势，并且酶活性也有相应提高。
根据本发明一种典型的实施方式，提供一种DNA分子。该DNA分子编码上述单加氧酶突变体。上述DNA编码得到的单加氧酶，提高了酶活性和酶的立体选择性，在催化硫醚类化合物或酮类化合物的单加氧反应中可以减少加入的酶量，降低后处理难度。
本发明的上述DNA分子还可以以“表达盒”的形式存在。“表达盒”是指线性或环状的核酸分子，涵盖了能够指导特定核苷酸序列在恰当宿主细胞中表达的DNA和RNA序列。一般而言，包括与目标核苷酸有效连接的启动子，其任选的是与终止信号和/或其他调控元件有效连接的。表达盒还可以包括核苷酸序列正确翻译所需的序列。编码区通常编码目标蛋白，但在正义或反义方向也编码目标功能RNA，例如反义RNA或非翻译的RNA。包含目标多核苷酸序列的表达盒可以是嵌合的，意指至少一个其组分与其至少一个其他组分是异源的。表达盒还可以是天然存在的，但以用于异源表达的有效重组形成获得的。
根据本发明一种典型的实施方式，提供一种重组质粒。该重组质粒含有上述任一种DNA分子。上述重组质粒中的DNA分子置于重组质粒的适当位置，使得上述DNA分子能够正确地、顺利地复制、转录或表达。
虽然本发明在限定上述DNA分子时所用限定语为“含有”，但其并不意味着可以在DNA序列的两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在DNA序列的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定将不能真实地覆盖这些情形。
本发明中所使用的术语“质粒”包括双链或单链线状或环状形式的任何质粒、粘粒、噬菌体或农杆菌二元核酸分子，优选为重组表达质粒，可以是原核表达质粒也可以是真核表达质粒， 但优选原核表达质粒，在某些实施方案中，重组质粒选自pET-22b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b(+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。更优选，上述重组质粒是pET-22b(+)。
根据本发明一种典型的实施方式，提供一种宿主细胞，宿主细胞含有上述任一种重组质粒。适用于本发明的宿主细胞包括但不仅限于原核细胞、酵母或真核细胞。优选原核细胞为真细菌，例如革兰氏阴性菌或革兰氏阳性菌。更优选原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。单加氧酶诱导表达最佳条件：25℃，0.2mM IPTG诱导16h。将突变质粒转化至大肠杆菌细胞内，然后通过超声破碎细胞的方法获得粗酶。
根据本发明一种典型的实施方式，提供一种单加氧酶突变体在催化硫醚类化合物或酮类化合物的单加氧反应中的应用。其中，单加氧酶为上述任一种单加氧酶突变体。由于本发明的上述单加氧酶突变体具有更高的酶催化活性，因而利用本发明的单加氧酶突变体进行工业生产不仅能够降低生产成本，而且所获得的手性更纯的产品。
在本发明一种典型的实施方式中，硫醚类化合物为
Figure PCTCN2019083051-appb-000005
在上述式中，R 1和R 2分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。另外，R 1和R 2可单独或两者互相结合形成取代或未被取代的环。R 1和R 2优选为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。作为芳基，可以列举苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基、吡咯基氧基等。作为烷基，可以列举甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基、环庚基等。作为芳烷基，可以列举苄基等。这些基团还可以进一步被取代，作为其取代基，可以列举卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基等。另外，环的形成也可以经由这些取代基。酮类化合物为
Figure PCTCN2019083051-appb-000006
在上述式中，R 3和R 4分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。另外，R 3和R 4可单独或两者互相结合形 成取代或未被取代的环。R 3和R 4优选为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基。作为芳基，可以列举苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基、吡咯基氧基等。作为烷基，可以列举甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基、环庚基等。作为芳烷基，可以列举苄基等。这些基团还可以进一步被取代，作为其取代基，可以列举卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基等。另外，环的形成也可以经由这些取代基。
反应式如下：
Figure PCTCN2019083051-appb-000007
在本发明一种典型的实施方式中，本发明的单加氧酶突变体应用于硫培南侧链合成中，反应式如下：
Figure PCTCN2019083051-appb-000008
本发明所获得的单加氧酶突变体，可用于硫培南侧链的合成，避免了放热反应，获得了高光学纯度的(R，R)-1-氧-3-羟基四氢噻吩(转化率＞99％,ee值95.0％)，使该化合物的工业生产成本得到大幅度降低，使得该酶在工业生产中具有更好的应用价值。
单加氧酶可为单加氧酶突变体的溶液、冻干粉、固定化酶或固定化细胞。
优选地，催化单加氧反应的辅因子为NAD +/NADH和/或NADP +/NADPH，辅因子循环系统包括于葡萄糖和葡糖脱氢酶、甲酸盐和甲酸脱氢酶、葡糖6-磷酸和葡糖-6-磷酸脱氢酶、仲醇(例如异丙醇)和/或仲醇脱氢酶及类似系统，最选地是用异丙醇和醇脱氢酶。
优选地，催化单加氧反应的温度为10～37℃，更优选为15～35℃；催化单加氧反应的时间为3～48h，更优选为6～16h；催化单加氧反应在pH为6.0～10.0的条件下进行，更优选pH为 6.0～9.0；此反应条件下，能够更好的发挥酶的催化性能。
下面将结合实施例进一步说明本发明的有益效果。
实施例1
单加氧酶定点突变制备(R，R)-1-氧-3-羟基四氢噻吩的反应特性比较
50mL的玻璃三角瓶中，将100mg(R)-3-羟基四氢噻吩加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL无水乙醇，12000rpm离心5min，上清加0.5g无水MgSO 4除水，12000rpm离心5min，取上清用N 2吹干后用700μL无水乙醇重新溶解，送GC分析。部分突变体反应特性如下表1：
表1
突变体 转化率 e.e.
野生型 + *
F508M ++ *
F508Y +++ **
F508N + **
F435S +++ **
F435N +++ *
F435A +++ *
F435Y +++ *
L147Y ++ **
L147T + **
L147M ++ **
L146F + **
L146Y + **
F280Y ++ *
L429Y ++ *
T436A +++ **
T436S ++ **
L438A ++ **
L438F ++ **
L438S ++ **
I494A ++ **
W493A ++ *
L510V ++ *
转化率在10％～50％的+，在50％～90％++，在90％以上的+++；ee值在-99％～-50％的*，在-50％～0％的**。
单点突变体的立体选择性和活性较母本都有所提高，但并未达到最理想的效果，因此对有益活性位点进行不同组合可以获得更优的突变体。
实施例2
单加氧酶组合突变制备(R，R)-1-氧-3-羟基四氢噻吩的反应特性比较
50mL的玻璃三角瓶中，将100mg(R)-3-羟基四氢噻吩加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL无水乙醇，12000rpm离心5min，上清加0.5g无水MgSO 4除水，12000rpm离心5min，取上清用N 2吹干后用700μL无水乙醇重新溶解，送GC分析。部分突变体反应特性如下表2：
表2
突变体 转化率 e.e.
野生型 + *
F435A+F508Y +++ ***
F435S+F508Y ++ **
L147Y+F508M ++ ***
F280Y+F508M ++ **
F280Y+F508N +++ **
F435A+L510V +++ ***
F435S+L510V ++ **
F435N+L510V +++ ***
T436A+L510V +++ ***
L438A+L510V +++ **
T436A+L438A +++ **
F435A+T436A +++ **
F435S+T436A +++ ***
F435N+T436A +++ **
L146Y+F508M ++ ***
L146F+F508M ++ **
F280Y+F508Y +++ **
F280Y+F508N ++ **
F435A+T436A+F508Y ++ **
F435A+T436A+F508M ++ **
F435A+T436A+L510V +++ **
F435S+T436A+L510V ++ ***
T436A+L438A+F508Y ++ **
T436A+L438A+F508M ++ **
T436A+L438A+F508N +++ ***
L147Y+F435A+F508M ++ ***
L147Y+F435A+F508Y ++ **
L147Y+F435A+F508N ++ *
L147Y+F435S+F508Y +++ **
L147Y+F435N+F508Y ++ **
L147Y+F435S+F508Y +++ ***
L147Y+F435N+F508Y +++ **
转化率在10％～50％的+，在50％～90％++，在90％以上的+++；ee值在-99％～-50％的*，ee值在-50％～0％的**，在0％～20％的***，在20％～60％的****,在60％～80％的*****,在80％～99％的******。
实施例3
单加氧酶饱和突变制备(R，R)-1-氧-3-羟基四氢噻吩的反应特性比较
50mL的玻璃三角瓶中，将100mg(R)-3-羟基四氢噻吩加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL无水乙醇，12000rpm离心5min，上清加0.5g无水MgSO 4除水，12000rpm离心5min，取上清用N 2吹干后用700μL无水乙醇重新溶解，送GC分析。部分突变体反应特性如下表3：
表3
Figure PCTCN2019083051-appb-000009
Figure PCTCN2019083051-appb-000010
转化率在10％～50％的活性为+，在50％～90％为++，在90％以上的为+++；ee值在-99％～-50％的*，在-50％～0％的**，在0％～20％的***，在20％～60％的****,在60％～80％的*****,在80％～99％的******。
通过进行迭代饱和突变，对立体选择性提高的突变位点进行叠加，获了ee值稳定提高的突变体；同时对初筛得到的活性位点氨基酸进行组合饱和突变，避免进化过程中，进化结果只限于局部最高点而不能到达全局最高点。最终获得立体选择性和活性最优的突变体。
实施例4
部分突变体制备(R，R)-1-氧-3-羟基四氢噻吩的反应特性比较
50mL的玻璃三角瓶中，将100mg(R)-3-羟基四氢噻吩加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL无水乙醇，12000rpm离心5min，上清加0.5g无水MgSO 4除水，12000rpm离心5min，取上清用N 2吹干后用700μL无水乙醇重新溶解，送GC分析。部分突变体反应特性如下表4：
表4
突变体 活性 e.e.
野生型 + *
F508Y+F435A+L438A+W49A+D60L +++ ****
F508Y+F435A+L438A+V144E +++ ****
F508Y+F435A+L438A+G145F +++ ****
F508Y+F435A+L438A+T436A+H167W +++ ****
F508Y+F435A+L438A+T436A+A169K +++ ****
F508Y+F435A+L438A+T436A+S179M +++ ****
F508Y+F435A+L438A+T436A+A246V +++ ****
F508Y+F435A+L438A+T436A+V247T +++ ****
F508Y+F435A+L438A+T436A+F284S +++ ****
F508Y+F435A+L438A+T436A+G285A +++ ****
F508M+F435A+L438A+T436A+T286A +++ ****
F508M+F435A+L438A+T436A+R330A +++ ****
F508M+F435A+L438A+T436A+L332R +++ ****
F508M+F435A+L438A+T436A+A328N +++ ****
F508Y+F435A+L438A+T436S+G382A +++ ****
F508Y+F435A+L438A+T436S+M427I +++ ****
F508Y+F435A+L438A+T436S+V428A +++ ****
F508Y+F435A+L438A+T436S+G430A +++ ****
F508Y+F435A+L438A+T436S+P431A +++ ****
F508Y+F435A+L438A+T436S+N432Y +++ ****
F508Y+F435A+L438A+T436S+G433Y +++ ****
F508Y+F435A+L438A+T436S+P434A +++ *****
F508Y+F435A+L438A+T436S+Y509M +++ *****
F508Y+F435A+L438A+T436S+G511L +++ *****
F508Y+F435A+L438A+T436S+G512I +++ *****
F508Y+F435A+L438A+T436S+L513V +++ *****
转化率在10％～50％的活性为+，在50％～90％为++，在90％以上的为+++；ee值在-99％～-50％的*，在-50％～0％的**，在0％～20％的***，在20％～60％的****,在60％～80％的*****,在80％～99％的******。
实施例5
单加氧酶制备(R，R)-1-氧-3-羟基四氢噻吩的应用
500mL的玻璃三角瓶中，将1g(R)-3-羟基四氢噻吩加入到8.5mL异丙醇中，混匀，调pH为6.0～9.0，滴加到含单加氧酶2g，异丙醇脱氢酶0.4g，500μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为100mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL无水乙醇，12000rpm离心5min，上清加0.5g无水MgSO 4除水，12000rpm离心5min，取上清用N 2吹干后用700μL无水乙醇重新溶解，送GC分析，反应结果如下表5：
表5
Figure PCTCN2019083051-appb-000011
转化率在10％～50％的活性为+，在50％～90％为++，在90％以上的为+++；ee值在-99％～-50％的*，在-50％～0％的**，在0％～20％的***，在20％～60％的****,在60％～80％的*****,在80％～99％的******；收率在0％～20％的为#，在20％～40％的为##，在40％～60％的为###。
实施例6
单加氧酶突变体对底物4-甲基环己酮的反应特性比较
50mL的玻璃三角瓶中，将100mg 4-甲基环己酮加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反应。16h后，取700μL反应体系，加1.4mL乙腈，12000rpm离心5min，取上清送HPLC分析。突变体反应特性如下表6：
表6
Figure PCTCN2019083051-appb-000012
转化率在10％～50％的活性为+，在50％～80％为++，在80％以上的为+++
实施例7
单加氧酶突变体对底物苯甲硫醚的反应特性比较
50mL的玻璃三角瓶中，将100mg苯甲硫醚加入到850μL异丙醇中，混匀，调pH为6.0～9.0，然后加到含单加氧酶800mg，异丙醇脱氢酶0.12g，50μL(20mg/mL)NADP +，0.1M Tris-HCl buffer的粗酶液中，总反应体积为10mL，体系pH为6.0～9.0，15℃～30℃恒温摇瓶反 应。16h后，取700μL反应体系，加1.4mL乙腈，12000rpm离心5min，取上清送HPLC分析。部分突变体反应特性如下表7：
表7
Figure PCTCN2019083051-appb-000013
转化率在10％～50％的活性为+，在50％～90％为++，在90％以上的为+++；ee值在0％～20％的*，在20％～60％的**,在60％～80％的***,在80％～99％的****。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (16)

  1. 一种单加氧酶突变体，其特征在于，所述单加氧酶突变体的氨基酸序列是由SEQ ID NO：1所示的氨基酸序列发生突变得到的氨基酸序列，所述突变至少包括如下突变位点之一：第49位、第60位、第61位、第144位、第145位、第146位、第147位、第167位、第169位、第189位、第246位、第247位、第280位、第284位、第285位、第286位、第287位、第328位、第330位、第332位、第382位、第427位、第428位、第429位、第430位、第431位、第432位、第433位、第434位、第435位、第436位、第438位、第441位、第493位、第494位、第508位、第509位、第510位、第511位、第512位和第513位，且所述突变为第49位的色氨酸突变为丙氨酸；第60位的天冬氨酸突变为亮氨酸；第61位的苏氨酸突变为谷氨酰胺；第144位的缬氨酸突变为谷氨酸；第145位的甘氨酸突变为苯丙氨酸；第146位的亮氨酸突变为苯丙氨酸或酪氨酸；第147位的亮氨酸突变为甲硫氨酸、苏氨酸或酪氨酸；第167位的组氨酸突变为色氨酸；第169位的丙氨酸突变为赖氨酸；第189位的丝氨酸突变为蛋氨酸；第246位的丙氨酸突变为缬氨酸；第247位的缬突变为苏氨酸；第280位的苯丙氨酸突变为酪氨酸、色氨酸或缬氨酸；第284位的苯丙氨酸突变为丝氨酸；第285位的甘氨酸突变为丙氨酸；第286位的苏氨酸突变为丙氨酸；第287位的苯丙氨酸突变为天冬氨酸；第328位的丙氨酸突变为天冬酰胺；第330位的精氨酸突变为丙氨酸；第332位的亮氨酸突变为精氨酸；第382位的甘氨酸突变为丙氨酸；第427位的蛋氨酸突变为异亮氨酸；第428位的缬氨酸突变为丙氨酸；第429位的亮氨酸突变为酪氨酸；第430位的甘氨酸突变为丙氨酸；第431位的脯氨酸突变为丙氨酸；第432位的天冬酰胺突变为酪氨酸；第433位的甘氨酸突变为酪氨酸；第434位的脯氨酸突变为丙氨酸；第435位的苯丙氨酸突变为丝氨酸、丙氨酸、天冬酰胺或酪氨酸；第436位的苏氨酸突变为丙氨酸、丝氨酸、甘氨酸、谷氨酸或半胱氨酸；第438位的亮氨酸突变为甘氨酸、丙氨酸、酪氨酸、苯丙氨酸或丝氨酸；第441位的丝氨酸突变为亮氨酸和缬氨酸；第493位的色氨酸突变为丙氨酸；第494位的异亮氨酸突变为丙氨酸、甲硫氨酸或丝氨酸，第508位的苯丙氨酸突变为酪氨酸、甲硫氨酸或天冬酰胺，第509位的酪氨酸突变为蛋氨酸；第510位的亮氨酸突变为缬氨酸；第511位的甘氨酸突变为亮氨酸；第512位的甘氨酸突变为异亮氨酸；第513位的亮氨酸突变为缬氨酸；或者所述单加氧酶突变体的氨基酸序列具有所述发生突变的氨基酸序列中的所述突变位点，且与所述发生突变的氨基酸序列具有80％以上同源性的氨基酸序列。
  2. 根据权利要求1所述的单加氧酶突变体，其特征在于，所述突变至少包括如下突变位点组合之一：F435A+F508Y；F435S+F508Y；L147Y+F508M；F280Y+F508M；F280Y+F508N；
    F435A+L510V；F435S+L510V；F435N+L510V；T436A+L510V；L438A+L510V；
    T436A+L438A；F435A+T436A；F435S+T436A；F435N+T436A；L146Y+F508M；
    L146F+F508M；F280Y+F508Y；F280Y+F508N；F435A+T436A+F508Y；
    F435A+T436A+F508M；F435A+T436A+L510V；F435S+T436A+L510V；
    T436A+L438A+F508Y；T436A+L438A+F508M；T436A+L438A+F508N；
    L147Y+F435A+F508M；L147Y+F435A+F508Y；L147Y+F435A+F508N；
    L147Y+F435S+F508Y；L147Y+F435N+F508Y；L147Y+F435S+F508Y；
    L147Y+F435N+F508Y；F508Y+F435A+L438A；F508Y+F435A+L438Y；
    F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；
    F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；
    F508Y+F435A+L438A+T436A+F280W；F508Y+F435A+L438A+T436A+F280A；
    F508Y+F435A+L438A+T436A+S441L；F508M+F435A+L438A+T436A+F280W；
    F508M+F435A+L438A+T436A+F280V；F508M+F435A+L438A+T436A+S441V；
    F508M+F435A+L438A+T436A+S441A；F508Y+F435N+L438A+T436S；
    F508Y+F435N+L438A+T436S+F280V；F508Y+F435N+L438A+T436S+S441L；
    F508Y+F435N+L438A+T436S+F280V+S441V；
    F508Y+F435N+L438A+T436S+F280V+S441V+L510V；
    F508M+F435N+L438A+T436S+F280V+S441V+L510V；
    F508M+F435A+L438A+T436S+F280V+S441V+L510V；
    F508M+F435S+L438A+T436S+F280V+S441V+L510V；
    F508Y+F435S+L438Y+T436S+F280V+S441V+L510V；
    F508Y+F435N+L438A+T436A+F280V+S441V+L510V；
    F508Y+F435N+L438A+T436A+F280V+S441A+L510V；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M；F508Y+F435N+L438A+T436S+F280V+S441V+L510V+D60L；
    F508M+F435N+L438A+T436S+F280V+S441V+L510V+D60L+T61Q；
    F508M+F435A+L438A+T436S+F280V+S441V+L510V+D60L+G145F；
    F508M+F435S+L438A+T436S+F280V+S441V+L510V+D60L+A169K；
    F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L+S189M；
    F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+L332R；
    F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L+A328N；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+G430A；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147M+I494A+D60L+N432Y；F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147Y+I494S+D60L+Y509M；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+L429Y+W493A+L146F+L147T+I494M+D60L+G512I；F508Y+F435A+L438A+W49A+D60L；F508Y+F435A+L438A+V144E；
    F508Y+F435A+L438A+G145F；F508Y+F435A+L438A+T436A+H167W；
    F508Y+F435A+L438A+T436A+A169K；F508Y+F435A+L438A+T436A+S179M；
    F508Y+F435A+L438A+T436A+A246V；F508Y+F435A+L438A+T436A+V247T；
    F508Y+F435A+L438A+T436A+F284S；F508Y+F435A+L438A+T436A+G285A；
    F508M+F435A+L438A+T436A+T286A；F508M+F435A+L438A+T436A+R330A；
    F508M+F435A+L438A+T436A+L332R；F508M+F435A+L438A+T436A+A328N；
    F508Y+F435A+L438A+T436S+G382A；F508Y+F435A+L438A+T436S+M427I；
    F508Y+F435A+L438A+T436S+V428A；F508Y+F435A+L438A+T436S+G430A；
    F508Y+F435A+L438A+T436S+P431A；F508Y+F435A+L438A+T436S+N432Y；
    F508Y+F435A+L438A+T436S+G433Y；F508Y+F435A+L438A+T436S+P434A；
    F508Y+F435A+L438A+T436S+Y509M；F508Y+F435A+L438A+T436S+G511L；
    F508Y+F435A+L438A+T436S+G512I；F508Y+F435A+L438A+T436S+L513V；
    F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+D60L；
    F508Y+F435N+L438A+T436A+F280V+S441V+L510V+D60L+P431A；
    F508Y+F435N+L438A+T436A+F280V+S441A+L510V+D60L；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147Y+I494S；
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+D60L+L429Y+W493A+L146F+L147T+I494M；F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；
    F508Y+F435A+L438A+T436S；F508M+F435A+L438A+T436A；
    F508M+F435A+L438A+T436S；F508Y+F435A+L438A；F508Y+F435A+L438Y；
    F508Y+F435A+L438Y；F508Y+F435A+L438A+T436A；F508Y+F435A+L438A+T436S；
    F508M+F435A+L438A+T436A；F508M+F435A+L438A+T436S；
    F508Y+F435A+L438A+T436A+F280W+D60L；
    F508Y+F435A+L438A+T436A+F280A+V247T；
    F508Y+F435A+L438A+T436A+S441L+F285A；F508Y+F435N+L438A+T436S+R330A；
    F508Y+F435N+L438A+T436S+F280V+G430A；
    F508Y+F435N+L438A+T436S+S441L+P434A；
    F508M+F435N+L438A+T436S+F280V+S441V+L510V+Q60L+T286A+Y509M；
    F508M+F435A+L438A+T436S+F280V+S441V+L510V+Q60L+T61Q+V247T；
    F508M+F435S+L438A+T436S+F280V+S441V+L510V+Q60L+F287D+R330A；
    F508Y+F435S+L438Y+T436S+F280V+S441V+L510V+V144E+G145F+M427I；
    F508Y+F435N+L438A+T436A+F280V+S441V+L510V+Q60L+L322R+N432Y；
    F508Y+F435N+L438A+T436A+F280V+S441A+L510V+R330A+P321A+G512I；和
    F508Y+F435N+L438A+T436A+F280V+S441L+L510V+T61Q+R330A+G430A。
  3. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1或2所述的单加氧酶突变体。
  4. 一种重组质粒，其特征在于，所述重组质粒含有权利要求3所述的DNA分子。
  5. 根据权利要求4所述的重组质粒，其特征在于，所述重组质粒为pET-22b(+)、pET-22b(+)、pET-3a(+)、pET-3d(+)、pET-11a(+)、pET-12a(+)、pET-14b(+)、pET-15b (+)、pET-16b(+)、pET-17b(+)、pET-19b(+)、pET-20b(+)、pET-21a(+)、pET-23a(+)、pET-23b(+)、pET-24a(+)、pET-25b(+)、pET-26b(+)、pET-27b(+)、pET-28a(+)、pET-29a(+)、pET-30a(+)、pET-31b(+)、pET-32a(+)、pET-35b(+)、pET-38b(+)、pET-39b(+)、pET-40b(+)、pET-41a(+)、pET-41b(+)、pET-42a(+)、pET-43a(+)、pET-43b(+)、pET-44a(+)、pET-49b(+)、pQE2、pQE9、pQE30、pQE31、pQE32、pQE40、pQE70、pQE80、pRSET-A、pRSET-B、pRSET-C、pGEX-5X-1、pGEX-6p-1、pGEX-6p-2、pBV220、pBV221、pBV222、pTrc99A、pTwin1、pEZZ18、pKK232-18、pUC-18或pUC-19。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求4或5所述的重组质粒。
  7. 根据权利要求6所述的宿主细胞，其特征在于，所述宿主细胞包括原核细胞、酵母或真核细胞；优选所述原核细胞为大肠杆菌BL21细胞或大肠杆菌DH5α感受态细胞。
  8. 一种如权利要求1或2所述的单加氧酶突变体在催化硫醚类化合物或酮类化合物的单加氧反应中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述硫醚类化合物为
    Figure PCTCN2019083051-appb-100001
    其中，R 1和R 2分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；R 1和R 2可单独或两者互相结合形成取代或未被取代的环；
    优选的，R 1和R 2为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；
    优选的，所述芳基包括苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基和吡咯基氧基；
    优选的，所述烷基包括甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基和环庚基；
    优选的，所述芳烷基为苄基；
    优选的，所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
  10. 根据权利要求8所述的应用，其特征在于，所述酮类化合物为
    Figure PCTCN2019083051-appb-100002
    其中，R 3和R 4分别独立的表示任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；R 3和R 4可单独或两者互相结合形成取代或未被取代的环；
    优选的，R 3和R 4为碳原子数1-20的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基，更优选的为碳原子数1-10的任选取代或未被取代的烷基、任选取代或未被取代的芳烷基、或任选取代或未被取代的芳基；
    优选的，所述芳基包括苯基、萘基、吡啶基、噻吩基、噁二唑基、咪唑基、噻唑基、呋喃基、吡咯基、苯氧基、萘氧基、吡啶基氧基、噻吩基氧基、噁二唑基氧基、咪唑基氧基、噻唑基氧基、呋喃基氧基和吡咯基氧基；
    优选的，所述烷基包括甲基、乙基、丙基、丁基、戊基、己基、异丙基、仲丁基、叔丁基、甲氧基、乙氧基、叔丁氧基、甲氧基羰基、乙氧基羰基、叔丁氧基羰基、乙烯基、烯丙基、环戊基和环庚基；
    优选的，所述芳烷基为苄基；
    优选的，所述取代是指被卤素原子、氮原子、硫原子、羟基、硝基、氰基、甲氧基、乙氧基、羧基、羧甲基、羧乙基或亚甲二氧基取代。
  11. 根据权利要求8所述的应用，其特征在于，所述应用为硫培南侧链合成。
  12. 根据权利要求8所述的应用，其特征在于，所述单加氧酶为权利要求1或2所述的单加氧酶突变体的溶液、冻干粉、固定化酶或固定化细胞。
  13. 根据权利要求8所述的应用，其特征在于，所述单加氧反应的反应体系中还包括辅因子，所述辅因子为NAD +/NADH和/或NADP +/NADPH，辅因子循环系统包括葡萄糖和葡糖脱氢酶、甲酸盐和甲酸脱氢酶、葡糖6-磷酸和葡糖-6-磷酸脱氢酶，或仲醇和仲醇脱氢酶。
  14. 根据权利要求8所述的应用，其特征在于，所述单加氧反应的温度为10～37℃，优选为15～35℃。
  15. 根据权利要求8所述的应用，其特征在于，所述单加氧反应的时间为3～48小时，更优选为6～16小时。
  16. 根据权利要求8所述的应用，其特征在于，所述单加氧反应在pH为5.0～10.0的条件下进行，优选pH为6.0～9.0。
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