WO2021077425A1 - 转氨酶突变体及其应用 - Google Patents

转氨酶突变体及其应用 Download PDF

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WO2021077425A1
WO2021077425A1 PCT/CN2019/113430 CN2019113430W WO2021077425A1 WO 2021077425 A1 WO2021077425 A1 WO 2021077425A1 CN 2019113430 W CN2019113430 W CN 2019113430W WO 2021077425 A1 WO2021077425 A1 WO 2021077425A1
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mtttefanreih
pet
amino acid
acid sequence
transaminase
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PCT/CN2019/113430
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French (fr)
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洪浩
詹姆斯·盖吉
肖毅
刘芳
张娜
王祖建
颜俊杰
史皖明
张慕姣
刘冶
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凯莱英医药化学（阜新）技术有限公司
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Priority to EP19949729.8A priority Critical patent/EP4050100A4/en
Priority to US17/755,266 priority patent/US20220380817A1/en
Priority to PCT/CN2019/113430 priority patent/WO2021077425A1/zh
Publication of WO2021077425A1 publication Critical patent/WO2021077425A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of biotechnology, in particular to a transaminase mutant and its application.
  • Optically pure amines and a- and b-amino acids play a key role in living organisms. These compounds are also very important in medical applications and are important intermediates for the synthesis of chiral drugs and natural products. There are significant differences in the pharmacological activity, metabolic process and toxicity of the enantiomers of chemical drugs containing chiral factors in the human body. The current research on chiral drugs has become one of the main directions of international new drug research.
  • Chiral amines are an important part of the synthesis of a variety of biologically active compounds and active pharmaceutical ingredients. It is estimated that 40% of current drugs are chiral amines and their derivatives, such as neurological drugs, cardiovascular drugs, and antihypertensive drugs. The synthesis of anti-infective drugs and vaccines all use chiral amines as intermediates (Top.Catal.2014,57,284-300), making chiral amine compounds an important part of the pharmaceutical industry.
  • Transaminase is a kind of biocatalyst with protein as the main body.
  • Transaminase uses pyridoxal phosphate as a cofactor, which can catalyze the transfer of an amino group on an amino donor (amino acid or amine) to a prochiral acceptor ketone to obtain a chiral amine or its by-product ketone or ⁇ -keto acid
  • the general term for the enzymes is a kind of biocatalyst with protein as the main body.
  • Transaminase uses pyridoxal phosphate as a cofactor, which can catalyze the transfer of an amino group on an amino donor (amino acid or amine) to a prochiral acceptor ketone to obtain a chiral amine or its by-product ketone or ⁇ -keto acid
  • the general term for the enzymes Because traditional chemical methods for asymmetric synthesis of amines have different limitations, such as low efficiency, low selectivity, and
  • transaminase The substrate spectrum of natural transaminase is often narrow and can only catalyze specific types of substrates to generate chiral amines. In the synthesis of biologically active compounds or active drugs, many types of chiral amines are required for synthesis. Therefore, This limits the wide application of transaminase.
  • industrial production processes often require certain organic solvents, pressure, pH and other conditions that are easy to denature proteins. Therefore, the biocatalysts used need to have high stability to meet the needs of industrial production.
  • wild transaminase is often inactivated under harsh conditions and has poor stability, which limits its wide application.
  • the present invention aims to provide a transaminase mutant and its application, so as to solve the technical problem that the SsTA protein has no catalytic activity on the carbonyl substrates with large steric hindrance in the prior art.
  • a transaminase mutant is provided.
  • the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO: 1.
  • the mutation includes at least one of the following mutation sites: G17V, L36P, Q40H, G69Y, H70T, L73A, V77G, V77S, V77T, A78I, Y130M, Y130V, Y130T, N132I, N132T, K141S, K142S, K142T, R143P, G144F, G144W, G144Y, E145D, E145S, E145G, K146R, L148A, L148I, L148T, L151A, L151H, L151H L151V, L151Q, T152R, H153P, H153G, Y158S, I160C, L163Q, L163R, A165I, P167S, E170R, T175A, P180A, Y198F, S207I, T290S, T290G, A292G, Y198M, R216Q, R216E, R216S, S217N, W217N F208R
  • the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO:1, and the mutation also includes at least one of the following mutation sites: T66M, T66A, T66Q, T66S, E105D, T134A , K150N, G187S, R188L, Y158N, I160F, I160L, I160V, Y162F, T204N, T204S, K211H, K211Q, L237Q, A242T, V244M, V244S, V244D, V244N, V244H, V244E, V244A, Y260F, Y260T270A, E261D , R273S, R273C, R273T, S278R, D327M and E282K; or the amino acid sequence of the amino acid sequence of the aminotransferase mutant has a mutation site in the mutated amino acid sequence, and an
  • the mutation includes at least one of the following mutation site combinations: L73A+V77G+T290S, H70T+L73A+V77G+A78I+K141S+R143P+S207I+T290S+A292G or H70T+L73A+V77G+G144F/Y/W +S207I.
  • the mutation includes at least one of the following mutation site combinations: G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G, G69Y+H70T+L73A+V77G+A78I+K141S+ K142S+R143P+G144F+S207I+T290S+A292G+A165I, G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198M, G69Y+HV77G+L73A+ A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198M, G69Y+HV77G+L73A+ A78I+K141S+K142S+R143P
  • a DNA molecule is provided.
  • This DNA molecule encodes the aforementioned transaminase mutant.
  • a recombinant plasmid is provided.
  • the recombinant plasmid contains DNA molecules.
  • the recombinant plasmids are pET-22a(+), 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-41
  • 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-DE3 cells or E. coli Rosetta-DE3 cells.
  • a method for producing chiral amines includes the step of catalyzing the transamination reaction of the ketone compound and the amino donor by the transaminase, and the transaminase is the above-mentioned mutant of the transaminase.
  • 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 form a substituted or unsubstituted ring alone or in combination with each other;
  • 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 to 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, Thienyloxy, oxadiazolyloxy, imidazolyloxy, thiazolyloxy, furyloxy and pyrrolyloxy;
  • the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, Methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
  • the aralkyl group is benzyl
  • the substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group. .
  • the ketone compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • amino donor is isopropylamine or alanine, preferably isopropylamine.
  • the pH is 7-11, preferably 8-10, and more preferably 9-10; preferably, the transaminase reacts to the ketone compound
  • the temperature of the reaction system for the catalytic transamination reaction with the amino donor is 25°C to 60°C, more preferably 30 to 55°C, and even more preferably 40 to 50°C.
  • the transaminase mutant of the present invention is based on the transaminase (SsTA protein) shown in SEQ ID NO: 1, and is mutated through site-directed mutagenesis, thereby changing its amino acid sequence to achieve changes in protein structure and function, and then through targeted mutations.
  • the screening method obtained the transaminase with the above-mentioned mutation sites. Therefore, the modified SsTA protein mutant obtained the catalytic activity on the carbonyl substrate, and the catalytic effect was better.
  • SsTA is a transaminase derived from Sciscionella sp., which can catalyze carbonyl substrates to generate chiral amines, but it cannot catalyze substrates with large steric hindrance, such as sitagliptin precursor ketone.
  • Sitagliptin precursor ketone has no catalytic activity in natural enzymes and modified enzymes due to its large steric hindrance.
  • ATA-117RD11 developed by Merck has this catalytic ability. Therefore, the development of SsTA mutants to obtain the catalytic ability of large-structure substrates, such as sitagliptin precursor ketone, is a challenging development.
  • the rational transformation of enzymes is based on the three-dimensional molecular structure of the enzyme to modify the substrate binding site, coenzyme binding site, surface and other parts of the enzyme to change the catalytic properties of the enzyme and improve the activity and selectivity of the enzyme.
  • the directed evolution of enzymes is a kind of irrational design of proteins, artificially creating special evolutionary conditions, simulating natural evolutionary mechanisms, modifying genes in vitro, applying error-prone PCR, DNA shuffling and other technologies, combined with efficient screening systems to obtain New enzymes with expected properties.
  • the technical scheme of the present invention rationally transforms the SsTA protein through a combination of rational design and random mutation technology, and the obtained mutant expands the substrate spectrum and obtains the catalytic ability for large-structure substrates; at the same time, its stability is enhanced, which can be used in The catalytic reaction is carried out under the conditions of high concentration of solvent and high temperature.
  • Rational design can be carried out by means of site-directed mutation.
  • site-directed mutagenesis refers to the introduction of desired changes (usually changes in a favorable direction), including bases, into the target DNA fragment (which can be a genome or a plasmid) by polymerase chain reaction (PCR) and other methods. Add, delete, point mutation, etc.
  • 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 to the template plasmid and then "circularly extended" by polymerase.
  • the so-called cyclic extension means that the polymerase extends the primers according to the template and returns after one turn. The 5'end of the primer is terminated, and then it undergoes repeated heating and annealing extension cycles. This reaction is different from rolling circle amplification and does not form multiple tandem copies.
  • the extension products of the forward and reverse primers are annealed and matched to become an open-circle plasmid with nicks.
  • the extension product of Dpn I digestion because the original template plasmid is derived from conventional E. coli, it is modified by dam methylation, and it is sensitive to Dpn I and is chopped up, while the plasmid with mutation sequence synthesized in vitro is not methylated. Instead of being cut, it can be successfully transformed in the subsequent transformation, and a clone of the mutant plasmid can be obtained.
  • the mutant plasmid must be transformed into E. coli cells and overexpressed in E. coli. Then, the crude enzyme is obtained by sonicating the cells. The best conditions for inducing expression of transaminase: 25°C, 0.1mM IPTG induction overnight.
  • the present invention transforms the SsTA protein, and carries out amino acid mutations on the protein through rational design and directed evolution, through (G17V, L36P, Q40H, G69Y, H70T, L73A, V77G, V77S, V77T, A78I, Y130M, Y130V, Y130T, N132I, N132T, K141S, K142S, K142T, R143P, G144F, G144W, G144Y, E145D, E145S, E145G, K146R, L148A, L148I, L148T, L151A, L151H, L151E, L151V, L151Q, T152R, H153158S, H153G, Y153G I160C, L163Q, L163R, A165I, P167S, E170R, T175A, P180A, Y198F, S207I, T290S, T290G, A292G, Y198M, R216Q,
  • ND means no product generation is detected
  • + means the conversion rate of the product obtained using 10wt wet cells is ⁇ 50%
  • -means the conversion rate of the product obtained using 10wt wet cells is ⁇ 10%
  • ++ means the conversion rate of the product obtained using 10wt wet cells>80%
  • +++ means using 3wt wet cells to obtain a product conversion rate of ⁇ 70%
  • ++++ means using 3wt wet cells to obtain a product conversion rate of >70%.
  • SsTA having as SEQ ID NO: amino acid sequence of 1, SEQ ID NO: 1MTTTEFANSNLVAVEPGAIREPTPPGSVIQYSEYELDRSQPLAGGVAWIEGEYVPADEARISIFDTGFGHSDLTYTVAHVWHGNIFRLEDHLDRLLHGAARLKLETGMSREELAGIAKRCVSLSQLREAYVNITITRGYGKKRGEKDLTKLTHQVYVYAIPYLWAFPPEEQIFGTSVIVPRHVRRAGRNTIDPTIKNYQWGDLTAASFEAKDRGARSAVLLDADNCVAEGPGFNVVLVKDGALVSPSRNALPGITRKTVYEIAAAKGIETMLRDVTSSELYEADELMAVTTAGGVTPITSLDGEQVGNGEPGPITVAIRDRFWALMDEPSSLIEAIDY.
  • the modified SsTA protein mutant obtained the catalytic activity for carbonyl substrates with a certain steric hindrance, and the catalytic effect was better. On this basis, further transformation was carried out, and the sterically hindered sitagliptin was selected before Body ketone is a living test substrate, and the catalytic activity of SsTA is continuously expanded through methods such as site-directed mutagenesis, saturation mutagenesis, and directed evolution. Before the activity verification, 300 natural aminotransferases and mutants including SsTA were tested for the activity of sitagliptin precursor ketone, and it was found that there was no catalytic activity, indicating that sitagliptin precursor ketone was greatly hindered due to steric hindrance.
  • ND means that no product production is detected when using >10wt wet cells in the catalytic reaction,-means that the conversion rate of the product obtained using 10wt wet cells is ⁇ 10%, + means that the conversion rate of the product obtained using 10wt wet cells is 10%-30%, ++ means Using 10wt wet cells to obtain a product conversion rate of 40% to 70%, +++ means using 10wt wet cells to obtain a product conversion rate of 70% to 90%, ++++ means using 10wt wet cells to obtain a product conversion rate of >90%, + ++++ means that the conversion rate of products obtained by using 5wt wet cells is >90%, and ++++++ means that the conversion rate of products obtained by using 3wt wet cells is >90%.
  • the amino acid sequence of "MTTTEFANREFH" and "MTTTEFANREIH” added at the N-terminus of SEQ ID NO:1.
  • Mutants modified by site-directed mutagenesis and directed evolution have acquired the ability to catalyze substrates with greater steric hindrance, and their vitality has been gradually improved.
  • the tolerance of the mutants was modified through error-prone PCR and directed screening methods.
  • the random mutation gene was constructed by error-prone PCR, and the gene containing the random mutation was constructed on the pET22b vector and transformed into BL21(DE3) host bacteria to construct a mutant strain library. After sequencing, the mutation frequency was adjusted to maintain about 1 to 3 amino acid mutations in each target protein.
  • the mutant library was induced to obtain proteins, and sitagliptin precursor ketone was used as the screening substrate, and the mutants were screened by setting different temperature and solvent tolerance conditions. The screened strains were sequenced and re-verified to confirm beneficial mutants. See Table 3 for details.
  • the tolerance-modified mutants were verified with sitagliptin precursor ketone as the substrate.
  • the optimal reaction temperature for M19 was 30°C, and the optimal reaction temperature for M38, M41 and other mutants was increased to 35°C.
  • the stability of the mutant in the solvent was also significantly improved, and the optimal reaction DMSO concentration was increased from 5% to 30%.
  • a transaminase mutant is provided.
  • the amino acid sequence of the transaminase mutant is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO: 1.
  • the mutation includes at least one of the following mutation sites: G17V, L36P, Q40H, G69Y, H70T, L73A, V77G, V77S, V77T, A78I, Y130M, Y130V, Y130T, N132I, N132T, K141S, K142S, K142T, R143P, G144F, G144W, G144Y, E145D, E145S, E145G, K146R, L148A, L148I, L148T, L151A, L151H, L151H L151V, L151Q, T152R, H153P, H153G, Y158S, I160C, L163Q, L163R, A165I, P167S, E170R, T175
  • the mutation includes at least one of the following mutation sites: T66M, T66A, T66Q, T66S, E105D, T134A, K150N, G187S, R188L, Y158N, I160F, I160L, I160V, Y162F, T204N, T204S, K211H, K211Q, L237Q , A242T, V244M, V244S, V244D, V244N, V244H, V244E, V244A, Y260F, Y260N, E261D, T270A, R273S, R273C, R273T, S278R, E282K, D327M; or the amino acid sequence of the transaminase mutant has the occurrence
  • the mutation site in the mutated amino acid sequence has an amino acid sequence that has more than 80% homology with the mutated amino acid sequence.
  • the transaminase mutant of the present invention is based on the transaminase (SsTA protein) shown in SEQ ID NO: 1, and is mutated through site-directed mutagenesis, thereby changing its amino acid sequence to achieve changes in protein structure and function, and then through targeted mutations.
  • the screening method obtained the transaminase with the above-mentioned mutation sites. Therefore, the modified SsTA protein mutant obtained the catalytic activity on the carbonyl substrate, and the catalytic effect was better.
  • the term "homology" used herein has the meaning generally known in the art, and those skilled in the art are also familiar with the rules and standards for determining the homology between different sequences.
  • the sequences defined by the present invention with different degrees of homology must also have improved resistance to organic solvents by the transaminase.
  • it is preferable that the amino acid sequence of the aminotransferase mutant has the above homology and has or encodes an amino acid sequence with improved resistance to organic solvents. Those skilled in the art can obtain such variant sequences under the teaching of the disclosure of this application.
  • the mutation includes at least one of the following mutation site combinations: L73A+V77G+T290S, H70T+L73A+V77G+A78I+K141S+R143P+S207I+T290S+A292G or H70T+L73A+V77G+G144F/Y/W+ S207I.
  • the mutation includes at least one of the following mutation site combinations: G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G, G69Y+H70T+L73A+V77G+A78I+K141S +K142S+R143P+G144F+S207I+T290S+A292G+A165I, G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198M, G69Y+HV77G+L73A+ +A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198M, G69Y+HV77G+L73A+ +A78I+K141S+K142S+R
  • a DNA molecule encodes the aforementioned transaminase mutant.
  • the above-mentioned transaminase mutants encoded by the DNA molecule obtained the catalytic activity for carbonyl substrates, and the catalytic effect was better, and some of the mutants also obtained the catalytic activity for sitagliptin precursor ketone, and improved the stability of the enzyme. And tolerance, reducing the amount of enzymes, reducing the difficulty of post-processing, making it suitable for industrial production.
  • 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 direction, 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 above-mentioned 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, phage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form. It is preferably a recombinant expression plasmid, which can be a prokaryotic expression plasmid or a prokaryotic expression plasmid. It can be a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid.
  • the recombinant plasmid is selected from pET-22a(+), 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 is provided, and the 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.
  • a method for producing a chiral amine includes the step of catalyzing the transamination reaction of the ketone compound and the amino donor by the transaminase, and the transaminase is any of the above-mentioned transaminase mutants. Because the above-mentioned transaminase mutants of the present invention have achieved catalytic activity on carbonyl substrates and have better catalytic effects, some of the mutants also obtained catalytic activity on sitagliptin precursor ketones, and improved enzyme stability And tolerance, reducing the amount of enzymes and reducing the difficulty of post-processing.
  • 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 may be alone or in combination 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 to 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, Thienyloxy, oxadiazolyloxy, imidazolyloxy, thiazolyloxy, furyloxy and pyrrolyloxy;
  • the alkyl group includes methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, sec-butyl, tert-butyl, methoxy, ethoxy, tert-butoxy, Methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, vinyl, allyl, cyclopentyl and cycloheptyl;
  • the aralkyl group is benzyl
  • the substitution refers to a halogen atom, a nitrogen atom, a sulfur atom, a hydroxyl group, a nitro group, a cyano group, a methoxy group, an ethoxy group, a carboxyl group, a carboxymethyl group, a carboxyethyl group or a methylenedioxy group. .
  • the ketone compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the amino donor is isopropylamine or alanine, preferably isopropylamine.
  • the pH is 7-11, preferably 8-10, more preferably 9-10, that is to say, the pH can be selected. It can optionally be a value from 7 to 11, such as 7, 7.5, 8, 8, 8.6, 9, 10, 10.5, and the like.
  • the temperature of the reaction system in which the transaminase catalyzes the transamination reaction of ketone compounds and amino donors is 25-60°C, more preferably 30-55°C, still more preferably 40-50°C, that is to say, the temperature can be any value.
  • the volume concentration of dimethyl sulfoxide in the reaction system where the transaminase catalyzes the transamination reaction of ketone compounds and amino donors is 0%-50%, for example, 10%, 15%, 18%, 20%, 30%, 35. %, 38%, 40%, 42%, 48%, 49%, etc.
  • the volume concentration of methyl tert-butyl ether in the reaction system where the transaminase catalyzes the transamination reaction of ketone compounds and amino donors is 0% to 90%, such as 10%, 16%, 18%, 20%, 30%, 35%, 38%, 40%, 42%, 48%, 49%, 55%, 60%, 70%, 80%, 90%, etc.
  • the results in Table 4 show that SsTA wild bacteria have no catalytic activity on the substrate, while the SsTA mutant can produce more than 90% of chiral amine products, and the chiral purity is >98% by ee value detection.
  • the SsTA mutant obtained the catalytic activity of the sterically hindered substrate sitagliptin precursor ketone, and the activity of some mutants was greatly improved, and the substrate spectrum was enlarged.
  • ND means no product generation is detected, + means 1%-10% product generation is detected, ++ means 10%-20% product generation is detected, +++ means 20%-30% product generation is detected, +++ + Means that 40%-50% of the product is detected.
  • ND no product generation is detected
  • +++ means 30%-40% product generation is detected
  • ++++ means 40%-50% product generation is detected.
  • ND no product generation is detected
  • +++ means 30%-40% product generation is detected
  • ++++ means 40%-50% product generation is detected.
  • ND no product generation is detected
  • +++ means 30%-40% product generation is detected
  • ++++ means 40%-50% product generation is detected.
  • the above-mentioned embodiments of the present invention achieve the following technical effects: the prior art SsTA wild bacteria have no catalytic activity on the substrate, and the modified mutant has an enlarged substrate spectrum. At the same time, the stability of the enzyme is improved, the optimal reaction temperature is significantly increased, and the solvent tolerance is improved, which can be applied to harsh reaction conditions.

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Abstract

本发明公开了一种转氨酶突变体及其应用。其中，该转氨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：G17V，L36P，Q40H，G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I等。本发明的转氨酶突变体获得了对羰基底物的催化活力。

Description

转氨酶突变体及其应用 技术领域
本发明涉及生物技术领域，具体而言，涉及一种转氨酶突变体及其应用。
背景技术
光学纯胺和a-和b-氨基酸在生物体中起着关键作用。这些化合物在医药应用中也非常重要，是合成手性药物和天然产物等的重要中间体。含手性因素的化学药物的对映体在人体内的药理活性、代谢过程及毒性存在显著的差异，当前手性药物的研究已成为国际新药研究的主要方向之一。
手性胺是合成多种生物活性化合物和活性药物成分的重要组成部分，据估计，目前40％的药物都是手性胺及其衍生物，如神经类药物、心血管药物、抗高血压药物、抗感染药物及疫苗等的合成都是以手性胺作为中间体(Top.Catal.2014,57,284-300)，使得手性胺化合物成为制药业的重要组成部分。
手性胺的工业生产有多种，主要依赖于烯酰胺从酮前体的金属催化氢化，过程需要昂贵的过渡金属配合物作为催化剂，由于这些过渡金属配合物资源有限，因此难以实现可持续性。同时，从酮前体经不对称合成手性胺的过程需要胺的保护和脱保护步骤，增加步骤和废物，降低收率。催化加氢还原法合成手性胺的方法中催化剂的制备困难而且价格昂贵，设备投资大，生产成本高，对催化剂活性和加氢条件要求很高，而且催化剂有毒性，尤其是氢气中的硫化物，极易发生人员中毒。
转氨酶是一类以蛋白质为主体的生物催化剂。转氨酶是以磷酸吡哆醛为辅因子，能够催化1个氨基供体(氨基酸或胺)上的氨基转移到前手性的受体酮，得到手性胺或其副产物酮或α-酮酸的酶类的总称。由于传统的不对称合成胺的化学方法存在不同的局限性，例如低效率，低选择性和对环境污染较严重等问题，同时转氨酶催化合成手性胺具有很高的立体和化学选择性，具有安全性和环境相容性，是绿色环保的过程，一步催化到位，具有化学方法不可比拟的优势，转氨酶合成手性化合物成为一种关键的不对称合成技术。
天然的转氨酶的底物谱往往较窄，只能催化特定类型的底物生成手性胺，而在生物活性化合物或活性药物合成过程中，需要多种类型的手性胺为原料进行合成，因此限制了转氨酶的广泛应用。同时，工业生产过程中往往需要在一定的有机溶剂、压力、pH等易使蛋白质变性的条件，因此需要所用的生物催化剂具有较高的稳定性，以适应工业化生产需要。而野生转氨酶往往在苛刻条件下较易失活，稳定性较差，从而限制了其广泛应用。
尽管使用转氨酶生产手性胺的进展已被高度关注，但酶促方法在制备手性胺化合物的应用中存在很多问题。如对空间位阻大的底物无催化活力；酶活性低及酶用量大导致发酵成本 增加；反应体系中稳定性不高易失活等。因此需要对天然转氨酶进行改造，扩大其底物谱，使其获得催化空间位阻较大的底物的能力，同时提高其稳定性，以提高其应用价值。
发明内容
本发明旨在提供一种转氨酶突变体及其应用，以解决现有技术中SsTA蛋白对空间位阻较大的羰基底物没有催化活力的技术问题。
为了实现上述目的，根据本发明的一个方面，提供了一种转氨酶突变体。该转氨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：G17V，L36P，Q40H，G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I，L148T，L151A，L151H，L151E，L151V，L151Q，T152R，H153P，H153G，Y158S，I160C，L163Q，L163R，A165I，P167S，E170R，T175A，P180A，Y198F，S207I，T290S，T290G，A292G，Y198M，R216Q，R216E，R216S，S217N，W200A，F208R，F208L和F208V；或者转氨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，转氨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，突变至少还包括如下突变位点之一：T66M，T66A，T66Q，T66S，E105D，T134A，K150N，G187S，R188L，Y158N，I160F，I160L，I160V，Y162F，T204N，T204S，K211H，K211Q，L237Q，A242T，V244M，V244S，V244D，V244N，V244H，V244E，V244A，Y260F，Y260N，E261D，T270A，R273S，R273C，R273T，S278R，D327M和E282K；或者转氨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，突变至少还包括如下突变位点组合之一：L73A+V77G+T290S，H70T+L73A+V77G+A78I+K141S+R143P+S207I+T290S+A292G或H70T+L73A+V77G+G144F/Y/W+S207I。
进一步地，突变至少包括如下突变位点组合之一：G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I +R216Q、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+R216S、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198F、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F208R、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+T204N、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F144W+Y198F、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198F+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+A165I+Y130M+163Q+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y198F+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+N132T+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148I+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+L151A+S207I+F208R+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+P180A+A165I+Y130M+L163Q+F208R+S207I+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y13 0M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+Y130V+K141S+K142T+R143P+G144F+E145D+L148T+L151A+A165I+LL163Q+F208R+S207I+K211H+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+Y130T+L163Q+A165I+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+S207I+T290S+A292G+A165I+Y130M+L163Q+K211H+F208R+L148A+L151A+T152R+M130V、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+L148A+L151A+P167S+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130V+L163Q+K211H+F208R+R188L+S207I+T290S+A292G、MTTTEFANREIH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+R188L+A165I+L163Q+Y130V+K211H+F208R+S207I+T290S+A292G+G17V+Q40H+T204S、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+R188L+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G+T204S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+E145S+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+K146R+E145G+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130M+163Q+L148A+L151E+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T204S+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+Y130M+K141S+K142T+R143P+G144Y+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、 MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151E+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+Y198F+T204S+F208R+S207I+K211H+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+T66A+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+G187S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+K211H+F208R+T204S+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+E105D+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+E145G+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151V+H153P+T152R+A165I+L163Q+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+V244M+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+I160F+L163Q+A165I+R188L+T204N+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+I160V+L163Q+A165I+R188L+T204S+S207 I+K211H+F208R+T290S+A292G、G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+K211H+F208R+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+Y260F+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+A242T+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+K150N+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T3S+Y260F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211Q+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F、 MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F+A242T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y162F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260N+E261D+F322Y、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T134A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T270A+E282K、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T175A+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151Q+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+L237Q、、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R14 3P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+D327M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+V244H、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R+R273T。
根据本发明的另一方面，提供了一种DNA分子。该DNA分子编码上述转氨酶突变体。
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DNA分子。
进一步地，重组质粒为pET-22a(+)、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。
根据本发明的又一方面，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-DE3细胞或大肠杆菌Rosetta-DE3细胞。
根据本发明的再一方面，提供了一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，转氨酶为上述转氨酶突变体。
进一步地，酮类化合物为
Figure PCTCN2019113430-appb-000001
转氨基反应产物为
Figure PCTCN2019113430-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，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。
优选的，酮类化合物为
Figure PCTCN2019113430-appb-000003
Figure PCTCN2019113430-appb-000004
进一步地，氨基供体为异丙胺或丙氨酸，优选为异丙胺。
进一步地，在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中，pH为7～11，优选为8～10，更优选为9～10；优选的，转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为25℃～60℃，更优选为30～55℃，进一步优选为40～50℃。本发明的转氨酶突变体是在SEQ ID NO：1所示的转氨酶(SsTA蛋白)的基础上，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转氨酶，因此改造后的SsTA蛋白突变体获得了对羰基底物的催化活力，且催化效果较好，在此基础上进一步进行改造，获得的突变体进一步验证对西他列汀前体酮的催化活力，后续进一步改造，提高了酶稳定性和耐受性，减少了酶用量，降低了后处理的难度，使得其能够适合工业化生产。
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。
SsTA是来源于Sciscionella sp.的转胺酶，可以催化羰基底物生成手性胺，但是其不能催化具有较大空间位阻的底物，如西他列汀前体酮。西他列汀前体酮由于其较大的空间位阻使得自然界中的天然酶及改造后的酶均无催化活力，目前只有默克公司开发的ATA-117RD11具 有此催化能力。因此，开发SsTA突变体，使其获得大结构底物的催化能力，如西他列汀前体酮等，是极具挑战的开发。
酶的理性改造是基于酶的三维分子结构对酶的底物结合部位、辅酶结合部位、表面及其他部位进行改造，以改变酶的催化特性，提高酶活力、选择性等特性。酶的定向进化是一种蛋白质的非理性设计，人为创造特殊的进化条件，模拟自然进化机制，在体外改造基因，应用易错PCR、DNA改组(DNA shuffling)等技术，结合高效筛选系统获得具有预期特性的新酶。
本发明技术方案通过理性设计和随机突变相结合的技术对SsTA蛋白进行理性改造，获得的突变体扩大了底物谱，获得了对大结构底物的催化能力；同时其稳定性增强，可在高浓度溶剂和高温度条件下进行催化反应。
理性设计可以通过定点突变的手段进行。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)，和模版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。Dpn I酶切延伸产物，由于原来的模版质粒来源于常规大肠杆菌，是经dam甲基化修饰的，对Dpn I敏感而被切碎，而体外合成的带突变序列的质粒由于没有甲基化而不被切开，因此在随后的转化中得以成功转化，即可得到突变质粒的克隆。
上述得将突变质粒转化至大肠杆菌细胞内，在大肠杆菌中过量表达。然后通过超声破碎细胞的方法获得粗酶。转氨酶诱导表达最佳条件：25℃，0.1mM IPTG诱导过夜。
通过采用软件对转氨酶的三维结构进行计算机模拟分析，发现G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I，L148T，L151A，L151H，L151E，L151V，L151Q，T152R，H153P，H153G，Y158S，I160C，L163Q，L163R，A165I，P167S，E170R，T175A，P180A，Y198F，S207I，T290S，T290G，A292G，Y198M，R216Q，R216E，R216S，S217N，W200A，F208R，F208L，F208V位于底物活性中性附近，其改变可有效扩增SsTA的底物结合口袋，从而使酶对底物的亲和性提高。T66M，T66A，T66Q，T66S，E105D，T134A，K150N，G187S，R188L，Y158N，I160F，I160L，I160V，Y162F，T204N，T204S，K211H，K211Q，L237Q，A242T，V244M，V244S，V244D，V244N，V244H，V244E，V244A，Y260F，Y260N，E261D，T270A，R273S，R273C，R273T，S278R，E282K，D327M可提高 酶的稳定性。
本发明对SsTA蛋白进行改造，通过理性设计和定向进化的方式对蛋白进行氨基酸突变，通过(G17V，L36P，Q40H，G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I，L148T，L151A，L151H，L151E，L151V，L151Q，T152R，H153P，H153G，Y158S，I160C，L163Q，L163R，A165I，P167S，E170R，T175A，P180A，Y198F，S207I，T290S，T290G，A292G，Y198M，R216Q，R216E，R216S，S217N，W200A，F208R，F208L，F208V)等突变来不断提高酶的催化活力，通过定向进化的方法对T66M，T66A，T66Q，T66S，E105D，T134A，K150N，G187S，R188L，Y158N，I160F，I160L，I160V，Y162F，T204N，T204S，K211H，K211Q，L237Q，A242T，V244M，V244S，V244D，V244N，V244H，V244E，V244A，Y260F，Y260N，E261D，T270A，R273S，R273C，R273T，S278R，E282K，D327M等位点进行改造，不断提高酶的稳定性。
为了扩大SsTA的底物谱及催化活力，选取如下底物为验活底物对获得的突变体进行活力验证，反应式如下：
Figure PCTCN2019113430-appb-000005
菌株信息及实验结果如表1所示。
表1
Figure PCTCN2019113430-appb-000006
Figure PCTCN2019113430-appb-000007
N.D.表示未检测到产品生成，+表示使用10wt湿细胞获得产品转化率<50％，-表示使用10wt湿细胞获得产品转化率<10％，++表示使用10wt湿细胞获得产品转化率>80％，+++表示使用3wt湿细胞获得产品转化率<70％，++++表示使用3wt湿细胞获得产品转化率>70％。
SsTA具有如SEQ ID NO：1所示的氨基酸序列，SEQ ID NO：1MTTTEFANSNLVAVEPGAIREPTPPGSVIQYSEYELDRSQPLAGGVAWIEGEYVPADEARISIFDTGFGHSDLTYTVAHVWHGNIFRLEDHLDRLLHGAARLKLETGMSREELAGIAKRCVSLSQLREAYVNITITRGYGKKRGEKDLTKLTHQVYVYAIPYLWAFPPEEQIFGTSVIVPRHVRRAGRNTIDPTIKNYQWGDLTAASFEAKDRGARSAVLLDADNCVAEGPGFNVVLVKDGALVSPSRNALPGITRKTVYEIAAAKGIETMLRDVTSSELYEADELMAVTTAGGVTPITSLDGEQVGNGEPGPITVAIRDRFWALMDEPSSLIEAIDY。
经改造后的SsTA蛋白突变体获得了对具有一定空间位阻的羰基底物的催化活力，且催化效果较好，在此基础上进一步进行改造，选取空间位阻极大的西他列汀前体酮为验活底物，通过定点突变、饱和突变、定向进化等方法，不断扩大SsTA的催化活力。在活力验证前，对包括SsTA在内的300种天然转氨酶及突变体进行西他列汀前体酮的活力验证，发现均无催化活力，说明西他列汀前体酮由于位阻极大使得天然的转氨酶对其均无催化活力，即使本实验室已有的突变体仍无催化活力。经改进一步造获得的SsTA突变体验证对西他列汀前体酮的催化活力。反应式如下：
Figure PCTCN2019113430-appb-000008
菌株信息及实验结果如表2所示。
表2
Figure PCTCN2019113430-appb-000009
Figure PCTCN2019113430-appb-000010
Figure PCTCN2019113430-appb-000011
Figure PCTCN2019113430-appb-000012
ND表示使用>10wt的湿细胞进行催化反应未检测到产品生成，-表示使用10wt湿细胞获得产品转化率<10％，+表示使用10wt湿细胞获得产品转化率10％～30％，++表示使用10wt湿细胞获得产品转化率40％～70％，+++表示使用10wt湿细胞获得产品转化率70％～90％，++++表示使用10wt湿细胞获得产品转化率>90％，+++++表示使用5wt湿细胞获得产品转化率>90％，++++++表示使用3wt湿细胞获得产品转化率>90％。“MTTTEFANREFH”和“MTTTEFANREIH”在SEQ ID NO：1的N端增加的氨基酸序列。
经过定点突变和定向进化改造的突变体获得了对具有较大空间位阻的底物的催化能力，且活力不断逐步提高。为了进一步提升突变体的稳定性性能，通过易错PCR和定向筛选的方法对突变体的耐受性进行改造。
经易错PCR构建获得随机突变基因，将含随机突变的基因构建到pET22b载体上，转化入BL21(DE3)宿主菌中，构建突变体菌株库。经测序鉴定调整突变频率使其保持在每个目的蛋白有约1～3个氨基酸突变。突变体库经诱导获得蛋白，以西他列汀前体酮为筛选底物，设置不同的温度和溶剂耐受条件对突变体进行筛选。筛选获得的菌株经测序及再次验证确认有益突变体，具体见表3。
表3
Figure PCTCN2019113430-appb-000013
Figure PCTCN2019113430-appb-000014
Figure PCTCN2019113430-appb-000015
+ 1经45℃，25％DMSO耐受处理1h后，残余活力提高0.1～1倍，++ 1表示经45℃，25％DMSO耐受处理1h后，残余活力提高1～2倍，+ 2表示经55℃，25％DMSO耐受处理1h后，残余活力提高1～2倍，++ 2表示经55℃，25％DMSO耐受处理1h后，残余活力提高2～3倍，+++ 2表示经55℃，25％DMSO耐受处理1h后，残余活力提高3～4倍。突变体经选择性测定ee值为95％～99％
经过耐受性改造的突变体以西他列汀前体酮为底物验证，M19最适反应温度为30℃，M38，M41等突变体的最适反应温度提升到35℃，M57，M58，M60，M72，M76最适反应温度提升到55℃。同时突变体在溶剂中稳定性也有显著提高，最适反应DMSO浓度由5％提升到30％。
根据本发明一种典型的实施方式，提供一种转氨酶突变体。该转氨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：G17V，L36P，Q40H，G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I，L148T，L151A，L151H，L151E，L151V，L151Q，T152R，H153P，H153G，Y158S，I160C，L163Q，L163R，A165I，P167S，E170R，T175A，P180A，Y198F，S207I，T290S，T290G，A292G，Y198M，R216Q，R216E，R216S，S217N，W200A，F208R，F208L和F208V。优选地，突变至少包括如下突变位点之一：T66M，T66A，T66Q，T66S，E105D，T134A，K150N，G187S，R188L，Y158N，I160F，I160L，I160V，Y162F，T204N，T204S，K211H，K211Q，L237Q，A242T，V244M，V244S，V244D，V244N，V244H，V244E，V244A，Y260F，Y260N，E261D，T270A，R273S，R273C，R273T，S278R，E282K，D327M；或者所述转氨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。
本发明的转氨酶突变体是在SEQ ID NO：1所示的转氨酶(SsTA蛋白)的基础上，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转氨酶，因此改造后的SsTA蛋白突变体获得了对 羰基底物的催化活力，且催化效果较好，在此基础上进一步进行改造，获得的突变体进一步验证对西他列汀前体酮的催化活力，后续进一步改造，提高了酶稳定性和耐受性，减少了酶用量，降低了后处理的难度，使得其能够适合工业化生产。
本文使用的术语“同源性”具有本领域通常已知的含义，本领域技术人员也熟知测定不同序列间同源性的规则、标准。本发明用不同程度同源性限定的序列还必须要同时具有改进的转氨酶对有机溶剂的耐受性。在上述实施方式中，优选转氨酶突变体的氨基酸序列具有以上的同源性并具有或编码具有改进的有机溶剂的耐受性的氨基酸序列。本领域技术人员可以在本申请公开内容的教导下获得这样的变体序列。
优选的，突变至少包括如下突变位点组合之一：L73A+V77G+T290S，H70T+L73A+V77G+A78I+K141S+R143P+S207I+T290S+A292G或H70T+L73A+V77G+G144F/Y/W+S207I.
更优选的，突变至少包括如下突变位点组合之一：G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+R216Q、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+R216S、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198F、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F208R、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+T204N、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F144W+Y198F、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198F+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+A165I+Y130M+163Q+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y198F+S2 07I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+N132T+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148I+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+L151A+S207I+F208R+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+P180A+A165I+Y130M+L163Q+F208R+S207I+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+Y130V+K141S+K142T+R143P+G144F+E145D+L148T+L151A+A165I+LL163Q+F208R+S207I+K211H+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+Y130T+L163Q+A165I+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+S207I+T290S+A292G+A165I+Y130M+L163Q+K211H+F208R+L148A+L151A+T152R+M130V、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+L148A+L151A+P167S+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130V+L163Q+K211H+F208R+R188L+S207I+T290S+A292G、MTTTEFANREIH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L 151A+T152R+R188L+A165I+L163Q+Y130V+K211H+F208R+S207I+T290S+A292G+G17V+Q40H+T204S、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+R188L+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G+T204S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+E145S+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+K146R+E145G+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130M+163Q+L148A+L151E+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T204S+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+Y130M+K141S+K142T+R143P+G144Y+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151E+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+Y198F+T204S+F208R+S207I+K211H+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+T66A+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+G187S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+K211H+F208R+T204S+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+E105D+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G14 4Y+E145G+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151V+H153P+T152R+A165I+L163Q+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+V244M+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+I160F+L163Q+A165I+R188L+T204N+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+I160V+L163Q+A165I+R188L+T204S+S207I+K211H+F208R+T290S+A292G、G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+K211H+F208R+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+Y260F+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+A242T+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208 R+K211H+T290S+A292G+K146R+E145G+K150N+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T3S+Y260F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211Q+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F+A242T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y162F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260N+E261D+F322Y、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260F、 MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T134A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T270A+E282K、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T175A+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151Q+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+L237Q、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+D327M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+V244H、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R+R273T。。
根据本发明一种典型的实施方式，提供一种DNA分子。该DNA分子编码上述转氨酶突变体。该DNA分子编码的上述转氨酶突变体获得了对羰基底物的催化活力，且催化效果较好，其中一些突变体还获得了对西他列汀前体酮的催化活力，并且提高了酶稳定性和耐受性，减少了酶用量，降低了后处理的难度，使得其能够适合工业化生产。
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-22a(+)、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。
根据本发明一种典型的实施方式，提供一种生产手性胺的方法。该方法包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，转氨酶为上述任一种转氨酶突变体。由于本发明的上述转氨酶突变体具有获得了对羰基底物的催化活力，且催化效果较好，其中一些突变体还获得了对西他列汀前体酮的催化活力，并且提高了酶稳定性和耐受性，减少了酶用量，降低了后处理的难度。
进一步地，酮类化合物为
Figure PCTCN2019113430-appb-000016
转氨基反应产物为
Figure PCTCN2019113430-appb-000017
其中，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。
优选的，酮类化合物为
Figure PCTCN2019113430-appb-000018
Figure PCTCN2019113430-appb-000019
在本发明一种典型的实施方式中，氨基供体为异丙胺或丙氨酸，优选为异丙胺。
应用本发明的转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中，pH为7～11，优选为8～10，更优选为9～10，也就是说pH的取值可以任选为7～11中的值，例如7、7.5、8、8、8.6、9、10、10.5等。转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为25～60℃，更优选为30～55℃，进一步优选为40～50℃，也就是说温度的取值可以任选为25～60℃中的值，例如30、31、32、35、37、38、39、40、42、45、48、50、51、52、55等。转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中二甲基亚砜体积浓度为0％～50％，例如选10％、15％、18％、20％、30％、35％、38％、40％、42％、48％、49％等。转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中甲基叔丁基醚体积浓度为0％～90％，例如选10％、16％、18％、20％、30％、35％、38％、40％、42％、48％、49％、55％、60％、70％、80％、90％等。
本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这 样的修改也落入本发明的范围。且下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例1
SsTA野生酶及突变体对西他列汀前体酮的催化活力
Figure PCTCN2019113430-appb-000020
100mL反应瓶中，称原料100mg，加入1mg 5’-磷酸吡哆醛，加入3.7mM异丙胺盐酸盐，加入1.5mLSsTA突变体或野生酶的粗酶液(0.2g突变体湿细胞经超声破碎制得20％粗酶液，pH＝8.0)，加入100mM PB8.0使体系终体积为5mL，与45℃恒温搅拌16h，体系与12000rpm离心5min后，取样200uL加入2mL乙腈溶解，于12000rpm离心5min后送HPLC检测产品转化率和ee值。表4结果可见SsTA野生菌对底物的无催化活力，而SsTA突变体可生成90％以上的手性胺产品，经ee值检测手性纯度>98％。经过本发明的改造后，SsTA突变体的获得了对空间位阻较大的底物西他列汀前体酮的催化活力，且部分突变体活力极大提高，扩大了底物谱。
表4
Figure PCTCN2019113430-appb-000021
Figure PCTCN2019113430-appb-000022
实施例2
SsTA野生酶及突变体催化酮化合物生成手性胺
Figure PCTCN2019113430-appb-000023
100mL反应瓶中，称原料100mg，加入1mg 5’-磷酸吡哆醛，加入3.7mM异丙胺盐酸盐，加入5mLSsTA突变体或野生酶的粗酶液(1g突变体湿细胞经超声破碎制得20％粗酶液，pH＝8.0)，加入100mM PB8.0使体系终体积为7mL，与30℃恒温搅拌16h，体系与12000rpm离心5min后，取样200uL加入2mL乙腈溶解，于12000rpm离心5min后送HPLC检测产品转化率。表5结果可见SsTA野生菌对底物的无催化活力，而SsTA突变体可生成40％以上的手性胺产品。经过本发明的改造后，SsTA突变体扩大了催化底物谱。
表5
Figure PCTCN2019113430-appb-000024
Figure PCTCN2019113430-appb-000025
ND表示未检测到产品生成，+表示检测到1％～10％产品生成，++表示检测到10％～20％产品生成，+++表示检测到20％～30％产品生成，++++表示检测到40％～50％产品生成。
实施例3
SsTA野生酶及突变体催化酮化合物生成手性胺
Figure PCTCN2019113430-appb-000026
100mL反应瓶中，称原料100mg，加入1mg 5’-磷酸吡哆醛，加入3.7mM异丙胺盐酸盐，加入5mLSsTA突变体或野生酶的粗酶液(1g突变体湿细胞经超声破碎制得20％粗酶液，pH＝8.0)，加入100mM PB8.0使体系终体积为7mL，与30℃恒温搅拌16h，体系与12000rpm 离心5min后，取样200uL加入2mL乙腈溶解，于12000rpm离心5min后送HPLC检测产品转化率。表6结果可见SsTA野生菌对底物的无催化活力，而SsTA突变体可生成40％以上的手性胺产品。经过本发明的改造后，SsTA突变体扩大了催化底物谱。
表6
Figure PCTCN2019113430-appb-000027
ND表示未检测到产品生成，+++表示检测到30％～40％产品生成，++++表示检测到40％～50％产品生成。
实施例4
SsTA野生酶及突变体催化酮化合物生成手性胺
Figure PCTCN2019113430-appb-000028
100mL反应瓶中，称原料100mg，加入1mg 5’-磷酸吡哆醛，加入3.7mM异丙胺盐酸盐，加入5mLSsTA突变体或野生酶的粗酶液(1g突变体湿细胞经超声破碎制得20％粗酶液，pH＝8.0)，加入100mM PB8.0使体系终体积为7mL，与30℃恒温搅拌16h，体系与12000rpm离心5min后，取样200uL加入2mL乙腈溶解，于12000rpm离心5min后送HPLC检测产品转化率。表7结果可见SsTA野生菌对底物的无催化活力，而SsTA突变体可生成40％以上的手性胺产品。经过本发明的改造后，SsTA突变体扩大了催化底物谱。
表7
Figure PCTCN2019113430-appb-000029
Figure PCTCN2019113430-appb-000030
ND表示未检测到产品生成，+++表示检测到30％～40％产品生成，++++表示检测到40％～50％产品生成。
实施例5
SsTA野生酶及突变体催化酮化合物生成手性胺
Figure PCTCN2019113430-appb-000031
100mL反应瓶中，称原料100mg，加入1mg 5’-磷酸吡哆醛，加入3.7mM异丙胺盐酸盐，加入5mLSsTA突变体或野生酶的粗酶液(1g突变体湿细胞经超声破碎制得20％粗酶液，pH＝8.0)，加入100mM PB8.0使体系终体积为7mL，与30℃恒温搅拌16h，体系与12000rpm离心5min后，取样200uL加入2mL乙腈溶解，于12000rpm离心5min后送HPLC检测产品转化率。表8结果可见SsTA野生菌对底物的无催化活力，而SsTA突变体可生成90％以上的手性胺产品，且手性纯度极高99％。经过本发明的改造后，SsTA突变体扩大了催化底物谱。
表8
Figure PCTCN2019113430-appb-000032
Figure PCTCN2019113430-appb-000033
ND表示未检测到产品生成，+++表示检测到30％～40％产品生成，++++表示检测到40％～50％产品生成。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：现有技术SsTA野生菌对底物无催化活力，经过改造后的突变体扩大的底物谱。同时改进了酶的稳定性，使其最适反应温度显著提高，溶剂耐受性提高，能适用于苛刻的反应条件。
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 (13)

  1. 一种转氨酶突变体，其特征在于，所述转氨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：G17V，L36P，Q40H，G69Y，H70T，L73A，V77G，V77S，V77T，A78I，Y130M，Y130V，Y130T，N132I，N132T，K141S，K142S，K142T，R143P，G144F，G144W，G144Y，E145D，E145S，E145G，K146R，L148A，L148I，L148T，L151A，L151H，L151E，L151V，L151Q，T152R，H153P，H153G，Y158S，I160C，L163Q，L163R，A165I，P167S，E170R，T175A，P180A，Y198F，S207I，T290S，T290G，A292G，Y198M，R216Q，R216E，R216S，S217N，W200A，F208R，F208L和F208V；或者所述转氨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所述的转氨酶突变体，其特征在于，所述转氨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，所述突变至少还包括如下突变位点之一：T66M，T66A，T66Q，T66S，E105D，T134A，K150N，G187S，R188L，Y158N，I160F，I160L，I160V，Y162F，T204N，T204S，K211H，K211Q，L237Q，A242T，V244M，V244S，V244D，V244N，V244H，V244E，V244A，Y260F，Y260N，E261D，T270A，R273S，R273C，R273T，S278R，D327M和E282K；或者所述转氨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。
  3. 根据权利要求1所述的转氨酶突变体，其特征在于，所述突变至少还包括如下突变位点组合之一：L73A+V77G+T290S，H70T+L73A+V77G+A78I+K141S+R143P+S207I+T290S+A292G或H70T+L73A+V77G+G144F/Y/W+S207I。
  4. 根据权利要求3所述的转氨酶突变体，其特征在于，所述突变至少包括如下突变位点组合之一：G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198M、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+R216Q、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+R216S、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+Y198F、 G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F208R、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+T204N、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+T290S+A292G+A165I+F144W+Y198F、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144W+S207I+T290S+A292G+A165I+Y198F+K211H、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+S207I+A165I+Y130M+163Q+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y198F+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+N132T+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+Y158S+I160C+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+L148I+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+A165I+Y130M+L163Q+L151A+S207I+F208R+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142S+R143P+G144F+P180A+A165I+Y130M+L163Q+F208R+S207I+K211H+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144F+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、G69Y+H70T+L73A+V77G+A78I+Y130V+K141S+K142T+R143P+G144F+E145D+L148T+L151A+A165I+LL163Q+F208R+S207I+K211H+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+Y130T+L163Q+A165I+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148 A+L151A+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+A165I+Y130M+L163R+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+S207I+T290S+A292G+A165I+Y130M+L163Q+K211H+F208R+L148A+L151A+T152R+M130V、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+L148A+L151A+P167S+A165I+Y130M+L163Q+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+A165I+Y130V+L163Q+K211H+F208R+R188L+S207I+T290S+A292G、MTTTEFANREIH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+R188L+A165I+L163Q+Y130V+K211H+F208R+S207I+T290S+A292G+G17V+Q40H+T204S、MTTTEFANREFH+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+L148A+L151A+T152R+R188L+A165I+Y130V+L163Q+K211H+F208R+S207I+T290S+A292G+T204S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+E145S+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141T+K142S+R143P+G144Y+K146R+E145G+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+A165I+Y130M+163Q+L148A+L151E+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T204S+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+Y130M+K141S+K142T+R143P+G144Y+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151E+T152R+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G、MTTTEFANREFH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+Y198F+T204S+F208R+S207I+K211H+T290S+A292G、 MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREFH+T66A+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+G187S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66Q+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+K211H+F208R+T204S+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+E105D+K141S+K142T+R143P+G144Y+Y130V+L148A+L151A+T152R+L163Q+A165I+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+E145G+A165I+Y130V+L163Q+L148A+L151A+T152R+R188L+T204S+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151V+H153P+T152R+A165I+L163Q+R188L+T204S+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+V244M+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+I160F+L163Q+A165I+R188L+T204N+S207I+K211H+F208R+T290S+A292G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130V+L148A+L151H+T152R+H153P+I160V+L163Q+A165I+R188L+T204S+S207I+K211H+F208R+T290S+A292G、G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+K146R+E145G+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+K211H+F208R+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R 188L+T204N+K211H+F208R+S207I+Y260F+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+A242T+T290S+A292G+S278R+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+K150N+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y158N、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273S、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T3S+Y260F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211Q+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204S+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+I160F+A242T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y162F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+ R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244E、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+V244A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260N+E261D+F322Y、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+Y260F、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T134A、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+S278R、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T270A+E282K、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+T175A+R273C、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151Q+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+S207I+F208R+K211H+T290S+A292G+K146R+E145G+L237Q、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+R273T、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+D327M、MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211H+F208R+S207I+T290S+A292G+S278R+V244H、 MTTTEFANREIH+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+K146R+E145G+L148A+L151H+T152R+H153P+L163Q+A165I+R188L+T204N+K211Q+F208R+S207I+T290S+A292G+S278R+R273T。
  5. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1至4中任一项所述的转氨酶突变体。
  6. 一种重组质粒，其特征在于，所述重组质粒含有权利要求5所述的DNA分子。
  7. 根据权利要求6所述的重组质粒，其特征在于，所述重组质粒为pET-22a(+)、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。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求6或7所述的重组质粒。
  9. 根据权利要求8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-DE3细胞或大肠杆菌Rosetta-DE3细胞。
  10. 一种生产手性胺的方法，包括转氨酶对酮类化合物及氨基供体进行催化转氨基反应的步骤，其特征在于，所述转氨酶为权利要求1至4中任一项所述的转氨酶突变体。
  11. 根据权利要求10所述的方法，其特征在于，所述酮类化合物为
    Figure PCTCN2019113430-appb-100001
    转氨基反应产物为
    Figure PCTCN2019113430-appb-100002
    其中，R1和R2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；R1和R2可单独或两者互相结合形成取代或未被取代的环；
    优选的，R1和R2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；
    优选的，所述酮类化合物为
    Figure PCTCN2019113430-appb-100003
    Figure PCTCN2019113430-appb-100004
  12. 根据权利要求10所述的方法，其特征在于，所述氨基供体为异丙胺或丙氨酸，优选为异丙胺。
  13. 根据权利要求10所述的方法，其特征在于，在转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系中，pH为7～11，优选为8～10，更优选为9～10；
    优选的，转氨酶对酮类化合物及氨基供体进行催化转氨基反应的反应体系的温度为25℃～60℃，更优选为30～55℃，进一步优选为40～50℃。
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