WO2023201809A1 - 转氨酶突变体及其应用 - Google Patents

转氨酶突变体及其应用 Download PDF

Info

Publication number
WO2023201809A1
WO2023201809A1 PCT/CN2022/093313 CN2022093313W WO2023201809A1 WO 2023201809 A1 WO2023201809 A1 WO 2023201809A1 CN 2022093313 W CN2022093313 W CN 2022093313W WO 2023201809 A1 WO2023201809 A1 WO 2023201809A1
Authority
WO
WIPO (PCT)
Prior art keywords
pet
transaminase
mutation
mutant
reaction
Prior art date
Application number
PCT/CN2022/093313
Other languages
English (en)
French (fr)
Inventor
洪浩
詹姆斯•盖吉
肖毅
张娜
焦学成
张克俭
王磊
赵军旗
凌君
徐新生
李�瑞
王祖建
Original Assignee
凯莱英医药集团（天津）股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 凯莱英医药集团（天津）股份有限公司 filed Critical 凯莱英医药集团（天津）股份有限公司
Publication of WO2023201809A1 publication Critical patent/WO2023201809A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Definitions

  • the present invention relates to the field of biotechnology, specifically to an aminotransferase mutant and its application.
  • Chiral amines refer to a class of compounds containing amino groups in the chiral center of small molecule compounds. They are key intermediates for many medicines and pesticides and have attracted more and more attention in pharmaceutical production and industrial and agricultural production. Among them, chiral amines are important intermediates for the synthesis of neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infectious drugs and vaccines. Currently, 40% to 50% of chiral drugs are chiral amine compounds. Efficient and convenient synthesis of chiral amine compounds is an important direction in organic synthetic chemistry research. For example, the diabetes treatment drug sitagliptin (Merck and Codexis) and the broad-spectrum contact herbicide glufosinate-ammonium (Bayer) both have chiral amine chemical modules.
  • the main methods for preparing chiral amines include chemical methods, biological resolution methods and biological asymmetric synthesis methods.
  • the chemical synthesis of chiral amines often requires the use of expensive metal catalysts and special solvents, which results in higher production costs, lower enantioselectivity, and will cause certain environmental pollution; while the enzymatic preparation of chiral aromatic amines has catalytic Characterized by high efficiency, strong stereoselectivity, mild reaction conditions, and environmental friendliness, it is in line with the general trend of green synthesis and has therefore become a hot spot in current research.
  • the enzymes currently used to prepare chiral aromatic amines are mainly lipases and aminotransferases.
  • ⁇ -aminotransferases ⁇ -TAs
  • ⁇ -TAs ⁇ -aminotransferases
  • Transaminase also known as aminotransferase, belongs to the class of transferases and can reversibly catalyze the amino transfer reaction between ketone groups and amino groups.
  • the enzyme is called alpha aminotransferase, otherwise it is called omega transaminase.
  • the products of alpha aminotransferase are generally only alpha amino acids, while omega transaminase can aminate keto acids, aldehydes and ketones, and has the characteristics of high stereoselectivity and reproducible cofactors. Therefore, omega transaminase is more widely used in the synthesis of medicines and pesticides. intermediate.
  • omega transaminase is more widely used in the synthesis of medicines and pesticides. intermediate.
  • only a few of the currently reported wild ⁇ -TAs can be directly used in the pharmaceutical industry, which are mainly limited by the instability of the enzyme and unfavorable reaction balance.
  • the present invention aims to provide a transaminase mutant and its application to improve the activity of transaminase.
  • an aminotransferase mutant is provided.
  • the transaminase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1, and the mutation at least includes: T291A; or the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and is consistent with the mutated amino acid sequence.
  • the amino acid sequence has more than 80% homology, preferably 85% homology, more preferably 90% homology, still more preferably 95% homology, still more preferably 99% homology. sex.
  • the mutation includes T291A and at least one of the following mutation sites: V17S, A56S, A56P, F64L, Y69L, Y69M, Y69C, Y69G, T70A, T70G, T70M, T70C, T70S, Y75F, G77E, G77S, G77V, G77T , G77L, I78H, T106S, M130I, M130L, N132W, N132A, T134I, T134A, T134V, Y139S, G140C, S141G, K142G, K142V, Y144I, Y144V, Y144P, Y144L, Y144M, Y1 44F, E145N, E145S, E145L, E145H , E145D, A148T, A148Q, H151A, H151D, H151Y, H151Q, R152K, R152T, Y158I, Y158V, I160G
  • the mutation includes at least one of the following mutation site combinations: T291A+S290L, T291A+S290V, T291A+S290T, T291A+G292A, T291A+G292L, T291A+G292I, T291A+G292D, T291A+G292A+Y75T, T291A +G292A+G77E, T291A+G292A+M130I, T291A+G292A+M130L, T291A+G292A+Y144S, T291A+G292A+Y144G, T291A+G292A+Y144I, T291A+G292A+Y144V, T2 91A+G292A+Y144P, T291A+G292A +Y144L, T291A+G292A+Y144H, T291A+G292A+Q163S, T291
  • T70C T291A+G292A+T70C+W164F , T291A+G292A+T70C+F233Y, T291A+G292A+T70C+I165V, T291A+G292A+T70C+Y144M+G145N, T291A+G292A+T70C+Y144M+G145S, T291A+G292A+T70C +G145L, T291A+G292A+T70C +Y69L ⁇ T291A+G292A+T70C+T142G ⁇ T291A+G292A+T70C+I160G ⁇ T291A+G292A+T70C+I160T ⁇ T291A+G292A+T70C+I160S ⁇ T291A+G292A+T70C+I160V ⁇ T 291A+G292A+T70C+I160V ⁇ T 291A+G292
  • T291A+G292A+T70C+I160G +S204L+V244S+T134I+M326Q T291A+G292A+T70C+I160G+S204L+V244S+T134I+M32 6Q+Y158V
  • T291A+G292A+T70C +I160G+S204L+V244S+T134I+M326Q+R216N T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216K
  • T291A+G292A+L232F T291A+G292A+F2 33W
  • T291A+G292A+F233Y T291A +G292A+T70G, T2
  • a DNA molecule is provided.
  • the DNA molecule encodes any of the transaminase mutants described above.
  • a recombinant plasmid contains any of the above-mentioned 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 the above recombinant plasmid.
  • the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is E. coli BL21-DE3 cell or E. coli Rosetta-DE3 cell; and the eukaryotic cell is yeast.
  • a method for producing chiral amines includes the step of catalyzing a transamination reaction of a ketone compound and an amino donor by an aminotransferase, and the transaminase is any one of the above transaminase mutants.
  • ketone compounds are wherein, R 1 is H, C1 ⁇ C10 alkyl which is unsubstituted or optionally substituted by halogen, C5 ⁇ C10 cycloalkyl which is unsubstituted or optionally substituted by halogen, C5 which is unsubstituted or optionally substituted by halogen.
  • R 2 is a C1 ⁇ C10 alkane optionally substituted by at least one group of H, halogen, unsubstituted or halogen, NH 3 or NH 3 with Boc or Cbz protection group
  • R 3 is a C6-12 aryl group that is unsubstituted or optionally substituted by halogen, a C3-6 heterocyclic group that is unsubstituted or optionally substituted by halogen; preferably, the ketone compound is
  • amino donor is isopropylamine or alanine, preferably isopropylamine.
  • the pH is 7 to 11, preferably 8 to 10.5, and more preferably 10.5; preferably, the transaminase catalyzes the transamination reaction of ketone compounds and amino groups.
  • the temperature of the reaction system in which the donor catalyzes the transamination reaction is 25 to 60°C, further preferably 30 to 55°C, and more preferably 50°C; preferably, the transaminase catalyzes the transamination reaction of the ketone compound and the amino donor.
  • the volume concentration of dimethyl sulfoxide in the reaction system is 0% to 50%.
  • the above-mentioned transaminase mutant of the present invention is based on the transaminase shown in SEQ ID NO: 1, and the mutant is obtained by carrying out amino acid mutation and combined mutation of the protein through rational design and directed evolution, thereby expanding the substrate of the enzyme Spectrum, improve the enzyme's catalytic ability for large sterically hindered substrates; at the same time enhance its stability, it can catalyze reactions under high concentration solvents and high temperature conditions, and the application of this mutant can increase the reaction rate and improve enzyme stability.
  • the dosage of enzyme is reduced and the difficulty of post-processing is reduced, making it suitable for industrial production; in addition, the stereoselectivity of the transaminase mutant has also been greatly improved, which can produce chiral amines more efficiently, reducing production costs and later stages. It is difficult to process and is suitable for industrial production of chiral amines.
  • Transaminase is a type of biocatalyst with protein as the main body.
  • certain organic solvents, pressure, temperature and other conditions that are easy to denature proteins are often required. Therefore, the biocatalyst used must have high tolerance. , to meet the needs of industrial production.
  • wild transaminase has low catalytic activity, narrow substrate spectrum, and often has low tolerance to industrial demand conditions, thus limiting its wide application.
  • the present invention strives to obtain mutants by conducting amino acid mutations and combination mutations on proteins through rational design and directed evolution, thereby expanding the substrate spectrum of the enzyme, improving the enzyme's catalytic ability for large sterically hindered substrates, and at the same time enhancing its stability. , can carry out catalytic reactions under high concentration solvents and high temperature conditions; and improve the stereoselectivity of (1) the chiral center in the structure.
  • the rational modification 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 enzyme activity, selectivity and other properties.
  • Directed evolution of enzymes is a kind of irrational design of proteins. It artificially creates special evolutionary conditions, simulates natural evolutionary mechanisms, transforms genes in vitro, applies error-prone PCR, DNA shuffling and other technologies, and combines with efficient screening systems to obtain New enzymes with expected properties.
  • the present invention conducts substrate 1 on 150 natural transaminase and 190 transaminase mutants existing in this laboratory. Activity screening revealed that only the transaminase mutant MTTTEFANREIH(12aa)+G17V+Q40H+T66M+G69Y+H70T+L73A+V77G+A78I+K141S+K142T+R143P+G144Y+Y130M+L148A+L151H+ originating from Sciscionella sp.
  • this application uses pET22b as an expression vector to perform site-directed mutation, saturation mutation, error-prone PCR, and combined mutation in the aminotransferase.
  • a mutation site was introduced into the vector to obtain a plasmid containing the mutant gene.
  • BL21 (DE3) as the expression strain, the mutant protein was obtained under the induction of IPTG. The mutants are then tested for activity and mutants with improved activity are selected.
  • site-directed mutagenesis refers to the introduction of desired changes (usually changes that represent a favorable direction) into the target DNA fragment (which can be a genome or a plasmid) through polymerase chain reaction (PCR) and other methods, including bases. Addition, deletion, point mutation, etc. Site-directed mutagenesis can quickly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful method in genetic research.
  • the method of introducing site-directed mutations using whole plasmid PCR is simple and effective, and it is a commonly used method at present.
  • the principle is that a pair of primers (forward and reverse) containing mutation sites are annealed to the template plasmid and then "cyclically extended" by polymerase.
  • the so-called cyclic extension means that the polymerase extends the primer according to the template and returns to the template after one cycle.
  • the 5' end of the primer is terminated and then undergoes repeated cycles of heating, annealing and extension. This reaction is different from rolling circle amplification and does not form multiple tandem copies.
  • the extension products of the forward and reverse primers anneal and pair to form a nicked open plasmid.
  • the extension product of Dpn I digestion is because the original template plasmid is derived from conventional E. coli and is modified by dam methylation. It is sensitive to Dpn I and is chopped. However, the plasmid with mutated sequence synthesized in vitro is not methylated. It is not cut, so it can be successfully transformed in the subsequent transformation, and the clone of the mutant plasmid can be obtained.
  • the mutant plasmid can be transformed into E. coli cells and overexpressed in E. coli.
  • the optimal conditions for transaminase induced expression 25°C, 0.1mM IPTG overnight.
  • the crude enzyme is then obtained by breaking the cells by sonication.
  • Reaction verification conditions Prepare substrate mother liquor (substrate dissolved in DMSO) and PLP mother liquor (10 mg pyridoxal 5-phosphate dissolved in 1 mL buffer). Add boric acid buffer, PLP mother liquor, and 6 M isozoic acid to the 10 mL reactor in sequence. Propylamine hydrochloride solution, substrate mother liquor, and finally enzyme (20% enzyme solution) were added, and the reaction was carried out in a shaking table at 200 rpm. After the reaction, acetonitrile was added to the reaction system, shaken to mix, and centrifuged at 12,000 rpm for 3 minutes. The supernatant was diluted. The appropriate multiples will be sent to HPLC for activity detection.
  • Reaction conditions A 5wt enzyme, PLP 2mg, DMSO 20%, IPN 30eq, 500V, 0.1M Tris-Cl9.0, 37°C, 40h;
  • N.D. means no product generation is detected
  • - means 0 to 1%
  • + means 1 to 5%
  • ++ means 5 to 10%
  • +++ means 10 to 15%
  • ++++ means >15%.
  • Reaction conditions B 2wt enzyme, PLP 4mg, DMSO 30%, IPN 30eq, 200V, 0.1M boric acid 10.0, 50°C, 16h;
  • Reaction conditions C 0.5wt enzyme, PLP 4g/L, DMSO 30%, IPN 30eq, 100V, 0.1M boric acid 10.0, 50°C, 16h;
  • Reaction conditions D 0.5wt enzyme, PLP 1g/L, DMSO 30%, IPN 30eq, 100V, 0.1M boric acid 10.5, 55°C, 16h;
  • Reaction conditions E 0.1wt enzyme, PLP 1g/L, DMSO 50%, IPN 5eq, 100V, 0.2M boric acid 10.5, 60°C, 16h;
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90% , +++++ means >90%.
  • the mutants after the above-mentioned tolerance modification have been verified by the enzyme activity of substrate 1.
  • the high-temperature stability of the mutants has been significantly improved.
  • the optimal reaction temperature of the Template is 37°C.
  • the mutants T291A+G292A+T70C and The optimal reaction temperature of mutants such as T291A+G292A+T70C+I160G is increased to 50°C.
  • ee value of transaminase mutant ((CP1+CP)-(CP2+CP3))/(CP+CP1+CP2+CP3)
  • Reaction conditions D 0.5wt enzyme, PLP 1g/L, DMSO 30%, IPN (isopropylamine hydrochloride) 30eq, 100V, 0.1M boric acid 10.5, 55°C, 16h;
  • Reaction conditions F 7wt enzyme, PLP 1g/L, DMSO 20%, IPN 30eq, 500V, 0.2M boric acid 10.5, 60°C, 16h;
  • Reaction conditions G 0.1wt enzyme, PLP 1g/L, DMSO 30%, IPN 10eq, 200V, 0.2M boric acid 10.5, 60°C, 16h;
  • Reaction conditions H 0.3wt enzyme, PLP 1g/L, DMSO 30%, IPN 10eq, 100V, 0.2M boric acid 10.5, 60°C, 16h;
  • Reaction conditions L 0.3wt enzyme, PLP 1g/L, DMSO 40%, IPN 10eq, 100V, 0.2M boric acid 10.5, 60°C, 16h;
  • Reaction conditions M 0.3wt enzyme, PLP 1g/L, DMSO 40%, IPN 10eq, 100V, 0.2M boric acid 10.5, 55°C, 16h;
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90% , +++++ means >90%;
  • N.D. means 0, * means 0 ⁇ 10%, ** means 10 ⁇ 50%, *** means 50 ⁇ 80%, **** means 80 ⁇ 90%, ***** means 90 ⁇ 95% , ****** means >95%.
  • mutants have been verified by the enzyme activity of substrate 1, and the selectivity of the mutants is gradually improving.
  • Mutants T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77L, T291A +G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292S transaminase mutant ee value increased to more than 70%, mutant T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T, T 291A +G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T, T 291A +G292A+T70C+I160G+S204L+V244S+T134I+M326Q+
  • a transaminase mutant is provided.
  • the amino acid sequence of the transaminase mutant is obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation at least includes: T291A; or the amino acid sequence of the transaminase mutant has the amino acid sequence of the mutation.
  • the mutation site, and the amino acid sequence having more than 80% homology with the mutated amino acid sequence preferably has 85% homology, more preferably has 90% homology, and further preferably has 95% The homology is more preferably 99%.
  • the mutation includes T291A and at least one of the following mutation sites: V17S, A56S, A56P, F64L, Y69L, Y69M, Y69C, Y69G, T70A, T70G, T70M, T70C, T70S, Y75F, G77E, G77S, G77V, G77T , G77L, I78H, T106S, M130I, M130L, N132W, N132A, T134I, T134A, T134V, Y139S, G140C, S141G, K142G, K142V, Y144I, Y144V, Y144P, Y144L, Y144M, Y1 44F, E145N, E145S, E145L, E145H , E145D, A148T, A148Q, H151A, H151D, H151Y, H151Q, R152K, R152T, Y158I, Y158V, I
  • the term "homology" used herein has a meaning commonly known in the art, and those skilled in the art are also familiar with the rules and standards for determining homology between different sequences. Sequences defined by varying degrees of homology of the present invention must also have improved tolerance of the transaminase to organic solvents. In the above embodiment, it is preferred that the amino acid sequence of the transaminase mutant has the above homology and has or encodes an amino acid sequence with improved tolerance to organic solvents. One skilled in the art can obtain such variant sequences based on the teachings of the present disclosure.
  • the mutation includes at least one of the following mutation site combinations: T291A+S290L, T291A+S290V, T291A+S290T, T291A+G292A, T291A+G292L, T291A+G292I, T291A+G292D, T291A+G292A+Y75T, T291A+ G292A+G77E, T291A+G292A+M130I, T291A+G292A+M130L, T291A+G292A+Y144S, T291A+G292A+Y144G, T291A+G292A+Y144I, T291A+G292A+Y144V, T29 1A+G292A+Y144P, T291A+G292A+ Y144L, T291A+G292A+Y144H, T291A+G292A+Q163S, T
  • a DNA molecule encodes the organic solvent-resistant transaminase mutant described above.
  • the above-mentioned transaminase mutant encoded by the DNA molecule has good organic solvent tolerance and high pH tolerance, and has high soluble expression characteristics and high activity characteristics.
  • DNA molecules of the present invention can also exist in the form of "expression cassettes".
  • "Expression cassette” refers to a linear or circular nucleic acid molecule, covering DNA and RNA sequences capable of directing the expression of a specific nucleotide sequence in an appropriate host cell. Generally, this includes a promoter operably linked to the target nucleotide, optionally operably linked to a termination signal and/or other regulatory elements.
  • the expression cassette may also include sequences required for correct 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.
  • An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components.
  • the expression cassette may also be naturally occurring but obtained by efficient recombinant formation for heterologous expression.
  • a recombinant plasmid is provided.
  • the recombinant plasmid contains any of the above-mentioned DNA molecules.
  • the DNA molecules in the recombinant plasmid are placed at appropriate positions in the recombinant plasmid so that the DNA molecules can be copied, 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, preferably a recombinant expression plasmid, which can be a prokaryotic expression plasmid or It can be a eukaryotic expression plasmid, but is 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 above recombinant plasmids.
  • Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells or eukaryotic cells.
  • the prokaryotic cells are eubacteria, such as Gram-negative bacteria or Gram-positive bacteria. More preferably, the prokaryotic cell is Escherichia coli BL21 cells or Escherichia coli DH5 ⁇ competent cells, and the eukaryotic cell is yeast.
  • a method for producing chiral amine includes the step of catalyzing a transamination reaction of a ketone compound and an amino donor by a transaminase, and the transaminase is any of the above-mentioned organic solvent-resistant transaminase mutants.
  • transaminase mutant of the present invention has an expanded enzyme substrate spectrum and higher catalytic ability for large sterically hindered substrates; at the same time, its stability is enhanced, and it can carry out catalytic reactions under high-concentration solvents and high-temperature conditions, and thus this mutation
  • the application of polymers can increase the reaction rate, improve enzyme stability, reduce enzyme dosage, and reduce the difficulty of post-processing, making it suitable for industrial production.
  • ketone compounds are wherein, R 1 is H, C1 ⁇ C10 alkyl which is unsubstituted or optionally substituted by halogen, C5 ⁇ C10 cycloalkyl which is unsubstituted or optionally substituted by halogen, C5 which is unsubstituted or optionally substituted by halogen.
  • R 2 is a C1 ⁇ C10 alkane optionally substituted by at least one group of H, halogen, unsubstituted or halogen, NH 3 or NH 3 with Boc or Cbz protection group
  • R 3 is a C6-12 aryl group that is unsubstituted or optionally substituted by halogen, or a C3-6 heterocyclic group that is unsubstituted or optionally substituted by halogen;
  • the ketone compound is (Substrate 1), (Substrate 2), (Substrate 3), (Substrate 4) or (Substrate 5).
  • the amino donor is isopropylamine or alanine, preferably isopropylamine.
  • the pH is 7 to 10.5, preferably 8 to 10, and more preferably 9 to 10. That is to say, the pH value can be It can optionally be a value from 7 to 10.5, such as 7, 7.5, 8, 8, 8.6, 9, 10, 10.5, etc.
  • the temperature of the reaction system in which transaminase catalyzes the transamination reaction of ketone compounds and amino donors is 25 to 60°C, more preferably 30 to 55°C, and further preferably 40 to 50°C.
  • the value of the temperature can be any Select a value from 25 to 60°C, such as 30, 31, 32, 35, 37, 38, 39, 40, 42, 45, 48, 50, 51, 52, 55, etc.
  • the volume concentration of dimethyl sulfoxide in the reaction system where transaminase catalyzes the transamination reaction of ketones and amino donors is 0% to 50%, for example, 10%, 15%, 18%, 20%, 30%, 35 %, 38%, 40%, 42%, 48%, 49%, etc.
  • Template and its mutants catalyze ketone compounds to generate chiral amines, and their activity was tested under reaction conditions M.
  • the results show that Template has poor catalytic activity on the substrate, while the Template mutant has better catalytic activity, and the mutant T291A+G292A +I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+T70G+G160T+T106S-12aa transaminase mutant ee value increased to more than 90%.
  • the Template mutants After the transformation of the present invention, the Template mutants have acquired catalytic activity for substrates with greater steric hindrance, the activity of some mutants has been greatly improved, and the stereoselectivity of the mutants has also been greatly improved, expanding the substrate spectrum. .
  • the reaction formula is as follows, and the results are shown in Table 4.
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90% , +++++ means >90%.
  • N.D. means 0, * means 0 ⁇ 10%, ** means 10 ⁇ 50%, *** means 50 ⁇ 80%, **** means 80 ⁇ 90%, ***** means 90 ⁇ 95% , ****** means >95%.
  • Template and its mutants catalyze ketone compounds to generate chiral amines, and their activity was tested under reaction conditions N. The results showed that Template has no catalytic activity on the substrate, while the Template mutant has better catalytic activity. After the transformation of the present invention, the Template mutant expands the catalytic substrate spectrum.
  • the reaction formula is as follows, and the results are shown in Table 5.
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90%, + ++++ means 90% to 95%, ++++++ means >95%.
  • Template and its mutants catalyze ketone compounds to generate chiral amines, and their activity was tested under reaction conditions N. The results showed that Template has no catalytic activity on the substrate, while the Template mutant has better catalytic activity. After the transformation of the present invention, the Template mutant expands the catalytic substrate spectrum.
  • the reaction formula is as follows, and the results are shown in Table 6.
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90%, + ++++ means 90% to 95%, ++++++ means >95%.
  • Template and its mutants catalyze ketone compounds to generate chiral amines, and their activity was tested under reaction conditions N. The results showed that Template has no catalytic activity on the substrate, while the Template mutant has better catalytic activity. After the transformation of the present invention, the Template mutant expands the catalytic substrate spectrum.
  • the reaction formula is as follows, and the results are shown in Table 7.
  • N.D. means no product generation is detected
  • - means 0 to 20%
  • ++ means 40 to 60%
  • +++ means 60 to 80%
  • ++++ means 80 to 90%
  • +++++ means 90 ⁇ 100%.
  • Template and its mutants catalyze ketone compounds to generate chiral amines, and their activity was tested under reaction conditions N. The results showed that Template has no catalytic activity on the substrate, while the Template mutant has better catalytic activity. After the transformation of the present invention, the Template mutant expands the catalytic substrate spectrum.
  • the reaction formula is as follows, and the results are shown in Table 8.
  • N.D. means no product generation is detected, - means 0 to 10%, + means 10 to 20%, ++ means 20 to 50%, +++ means 50 to 80%, ++++ means 80 to 90% , +++++ means >90%.
  • the mutant of the present invention expands the substrate spectrum of the enzyme and improves the enzyme's catalytic ability for large sterically hindered substrates; at the same time, it enhances In order to improve its stability, catalytic reactions can be carried out under high concentration solvents and high temperatures.
  • the application of this mutant can increase the reaction rate, improve enzyme stability, reduce the amount of enzyme, and reduce the difficulty of post-processing, making it possible Suitable for industrial production.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种转氨酶突变体及其应用。其中，该转氨酶突变体是由SEQ ID NO：1所示的氨基酸序列发生突变得到，突变至少包括：T291A；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，优选具有85％的同源性，更优选具有90％的同源性。该转氨酶突变体扩大了酶的底物谱、提高了立体选择性，提高酶对大位阻底物的催化能力；同时增强其稳定性，可在高浓度溶剂和高温度条件下进行催化反应生产手性胺。

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
手性胺是指小分子化合物手性中心含有氨基的一类化合物，是众多医药及农药的关键中间体，在药品生产和工农业生产中越来越受到人们的重视。其中，手性胺是合成神经类药物、心血管药物、抗高血压药物、抗感染药物及疫苗等的重要中间体，目前，40％～50％的手性药物都是手性胺类化合物，高效、便捷合成手性胺化合物是有机合成化学研究的重要方向。例如，糖尿病类治疗药物-西他列汀(Merck公司及Codexis公司)及广谱触杀型除草剂-草铵膦(Bayer公司)都具有手性胺化学模块。
目前，制备手性胺的方法主要有化学法、生物拆分法和生物不对称合成法。其中，化学法合成手性胺往往需要使用昂贵的金属催化剂及特制的溶剂，生产成本较高，对映选择性较低，并会造成一定的环境污染；而酶法制备手性芳香胺具有催化效率高、立体选择性强、反应条件温和、环境友好等特点，符合绿色合成的大趋势，因而成为当前研究的热点。目前用于制备手性芳香胺的酶主要为脂肪酶和转氨酶。相对于生物催化剂(脂肪酶)进行手性拆分法制备高纯度手性胺的最大理论收率只有50％，生物不对称合成法(转氨酶)理论收率高达100％，因此其在生产工业领域有着强大的应用前景，相关研究受到了广泛的重视。其中ω-转氨酶(ω-TAs)具有对映选择性和区域选择性高、底物谱较广以及无需额外添加昂贵的辅酶的优势，成为工业上用于生产手性胺的重要工业酶之一。
转氨酶又称氨基转移酶，属于转移酶类，能够可逆催化酮基与氨基之间的氨基转移反应。当这类酶催化的转氨反应中，底物或产物含有α氨基酸时，就称该酶为α转氨酶，反之则称之为ω转氨酶。α转氨酶的产物一般只是α氨基酸，而ω转氨酶能够氨基化酮酸、醛和酮，且具有立体选择性高、辅因子可再生的特点，因此，ω转氨酶被更广泛地应用于合成医药和农药中间体。但是目前报道的野生ω-TAs中能直接用于制药工业的为数不多，主要受限于酶的不稳定性及不利的反应平衡。
发明内容
本发明旨在提供一种转氨酶突变体及其应用，以提高转氨酶的活性。
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。该转氨酶突变体是由SEQ ID NO：1所示的氨基酸序列发生突变得到，突变至少包括：T291A；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，优选具有85％的同源性，更优选具有90％的同源性，进一步优选具有95％的同源性，再优选具有99％的同源性。
进一步地，突变包括T291A和至少如下突变位点之一：V17S、A56S、A56P、F64L、Y69L、Y69M、Y69C、Y69G、T70A、T70G、T70M、T70C、T70S、Y75F、G77E、G77S、G77V、G77T、G77L、I78H、T106S、M130I、M130L、N132W、N132A、T134I、T134A、T134V、Y139S、G140C、S141G、K142G、K142V、Y144I、Y144V、Y144P、Y144L、Y144M、Y144F、E145N、E145S、E145L、E145H、E145D、A148T、A148Q、H151A、H151D、H151Y、H151Q、R152K、R152T、Y158I、Y158V、I160G、I160T、I160S、I160V、I160C、I160T、Q163S、Q163T、Q163N、Q163A、W164F、I165L、I165H、I165A、I165V、P167A、P167K、Q171T、W200A、S204L、S204M、R216E、R216K、R216N、G232F、F233Y、A242S、A242Q、V244G、V244R、V244Y、V244S、V244K、V244M、T270G、T270H、S278L、S278R、S278K、E282C、E282R、E282K、T291A、T291Q、T291C、T291S、T291M、T291T、G292A、G292S和M326Q。
进一步地，突变至少还包括如下突变位点组合之一：T291A+S290L、T291A+S290V、T291A+S290T、T291A+G292A、T291A+G292L、T291A+G292I、T291A+G292D、T291A+G292A+Y75T、T291A+G292A+G77E、T291A+G292A+M130I、T291A+G292A+M130L、T291A+G292A+Y144S、T291A+G292A+Y144G、T291A+G292A+Y144I、T291A+G292A+Y144V、T291A+G292A+Y144P、T291A+G292A+Y144L、T291A+G292A+Y144H、T291A+G292A+Q163S、T291A+G292A+Q163T、T291A+G292A+Q163L、T291A+G292A+Q163N、T291A+G292A+Q163V、T291A+G292A+W164F、T291A+G292A+W164R、T291A+G292A+W164L、T291A+G292A+W164T、T291A+G292A+I165A、T291A+G292A+I165V、T291A+G292A+I165L、T291A+G292A+W200G、T291A+G292A+T70C、T291A+G292A+T70C+W164F、T291A+G292A+T70C+F233Y、T291A+G292A+T70C+I165V、T291A+G292A+T70C+Y144M+G145N、T291A+G292A+T70C+Y144M+G145S、T291A+G292A+T70C+G145L、T291A+G292A+T70C+Y69L、T291A+G292A+T70C+T142G、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160T、T291A+G292A+T70C+I160S、T291A+G292A+T70C+I160V、T291A+G292A+T70C+I160C、T291A+G292A+T70C+S204L、T291A+G292A+T70C+S204M、T291A+G292A+T70C+V244G、T291A+G292A+T70C+V244R、T291A+G292A+T70C+V244Y、T291A+G292A+T70C+V244S、T291A+G292A+T70C+V244K、T291A+G292A+T70C+V244M、T291A+G292A+T70C+I160G+H151A、T291A+G292A+T70C+I160G+H151D、T291A+G292A+T70C+I160G+H151Y、T291A+G292A+T70C+I160G+H151Q、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160G+S204L、T291A+G292A+T70C+I160G+V244S、T291A+G292A+T70C+I160G+S204L+V244S、T291A+G292A+T70C+I160G+T134I、T291A+G292A+T70C+I160G+A242S、T291A+G292A+T70C+I160G+A242Q、T291A+G292A+T70C+I160G+T270G、T291A+G292A+T70C+I160G+T270H、T291A+G292A+T70C+I160G+S278L、T291A+G292A+T70C+I160G+S278R、 T291A+G292A+T70C+I160G+S278K、T291A+G292A+T70C+I160G+E282C、T291A+G292A+T70C+I160G+E282R、T291A+G292A+T70C+I160G+E282K、T291A+G292A+T70C+I160G+M326Q、T291A+G292A+T70C+I160G+V17S、T291A+G292A+T70C+I160G+A56S、T291A+G292A+T70C+I160G+F64L、T291A+G292A+T70C+I160G+P167A、T291A+G292A+T70C+I160G+P167K、T291A+G292A+T70C+I160G+R216E、T291A+G292A+T70C+I160G+R216K、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216N、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216K、T291A+G292A+L232F、T291A+G292A+F233W、T291A+G292A+F233Y、T291A+G292A+T70G、T291A+G292A+T70M、T291A+G292A+T70A、T291A+G292A+T70S、T291A+G292A+T70C、T291A+G292A+W164Y、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+G77V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+N132A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77S、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+Y144F、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144W-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144P-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+N132R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165H-12aa、 T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+I78V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A291I-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+I78H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+T142V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145D-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+F233Y-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145D-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S2 78R+Y69M+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69G-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70S+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+S141G-12aa、T291A+G292A+T70A+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+Y139S-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+S292A-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+A291T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+I134V-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+T106S-12aa、或T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+Q171T-12aa。
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。该重组质粒含有上述任一种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、pKK232-8、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重组质粒。
进一步地，宿主细胞包括原核细胞或真核细胞；优选原核细胞为大肠杆菌BL21-DE3细胞或大肠杆菌Rosetta-DE3细胞；真核细胞为酵母。
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，转氨酶为上述任一种转氨酶突变体。
进一步地，酮类化合物为
Figure PCTCN2022093313-appb-000001
其中，R 1为H、未被取代或被卤素任选取代的C1～C10烷基、未被取代或被卤素任选取代C5～C10环烷基、未被取代或被卤素任选取代的C5～C10芳基或C5～C10杂芳基，R 2为H、卤素、未被取代或被卤素、NH 3或带Boc、Cbz保护的NH 3中至少一个基团所任选取代C1～C10烷基，R 3为未被取代或被卤素任选取代的C6-12的芳基、未被取代或被卤素任选取代的C3-6的杂环基；优选的，酮类化合物为
Figure PCTCN2022093313-appb-000002
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8～10.5，更优选为10.5；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℃，进一步优选为30～55℃，更优选为50℃；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中二甲基亚砜体积浓度为0％～50％。
本发明的上述转氨酶突变体是在SEQ ID NO：1所示的转氨酶的基础上，通过理性设计和定向进化的方式对蛋白进行氨基酸突变及其组合突变获得突变体，以此扩大酶的底物谱、提高酶对大位阻底物的催化能力；同时增强其稳定性，可在高浓度溶剂和高温度条件下进行催化反应，进而此突变体的应用可以提高反应速率，提高酶稳定性，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；另外，转氨酶突变体的立体选择性也得到了极大的提高，可以更加高效生产手性胺，降低了生产成本和后期处理难度，适合推广用于手性胺的工业生产。
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。
转氨酶是一类以蛋白质为主体的生物催化剂，而在工业生产过程中往往需要在一定的有机溶剂、压力、温度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耐受性，以适应工业化生产需要。而野生转氨酶一方面催化活性较低、底物谱较窄，且往往其对工业需求条件耐受性较低，从而限制了其广泛应用。
本发明力图通过理性设计和定向进化的方式对蛋白进行氨基酸突变及其组合突变获得突变体，以此扩大酶的底物谱、提高酶对大位阻底物的催化能力；同时增强其稳定性，可在高浓度溶剂和高温度条件下进行催化反应；并且提高结构中(1)手性中心的立体选择性。
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。
本发明对本实验室已有的150种天然转氨酶及190种转氨酶突变体进行底物1
Figure PCTCN2022093313-appb-000003
的活力筛选，发现只有来源于Sciscionella sp.的转氨酶突变体MTTTEFANREIH(12aa)+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(Template，其氨基酸序列为SEQ ID NO.1：MTTTEFANREIH(12aa)MTTTEFANSNLVAVEPVAIREPTPPGSVIQYSEYELDRSHPLAGGVAWIEGEYVPADEARISIFDMGFYTSDATYTGIHVWHGNIFRLEDHLDRLLHGAARLKLETGMSREELAGIAKRCVSLSQLREAMVNITITRGYGSTPYGRDATKHRPQVYVYAIPYQWIFPPEEQIFGTSVIVPRHVRRAGLNTIDPTIKNFQWGDLSAAIREAHDRGARSAVLLDADNCVAEGPGFNVVLVKDGALVSPSRNALPGITRKTVYEIAAAKGIETMLRDVTSSELYEADELMAVSTGGGVTPITSLDGEQVGNGEPGPITVAIRDRFWALMDEPSSLIEAIDY*(注，本申请中氨基酸位置编号 仍然对应Sciscionella sp.的转氨酶的位置编号，即SEQ ID NO.1中第1位氨基酸为12aa后的第1位“M”，依次类推)；对应的，Template碱基序列为SEQ ID NO.2：ATGACCACCACCGAGTTTGCCAACAGGGAATTCCAT(12aa)ATGACCACCACCGAGTTTGCCAACAGCAATCTGGTGGCCGTGGAACCGGTTGCAATCCGTGAGCCTACCCCTCCGGGCAGCGTGATTCAGTACAGCGAGTACGAACTGGATCGCAGCCATCCGCTGGCAGGTGGCGTTGCCTGGATTGAAGGCGAGTATGTTCCTGCCGATGAAGCCCGTATCAGCATCTTCGATATGGGCTTTTATACCAGCGATGCGACCTATACCGGGATCCACGTTTGGCACGGCAATATCTTCCGCCTGGAAGACCACCTGGACCGCCTGCTGCATGGTGCCGCACGTCTGAAACTGGAAACCGGTATGAGCCGCGAAGAACTGGCCGGCATTGCCAAGCGTTGTGTTAGCCTGAGCCAGCTGCGCGAAGCCATGGTGAACATCACCATTACCCGCGGCTATGGCAGCACCCCCTACGGAAGGGATGCGACCAAACATAGGCCCCAGGTGTACGTGTATGCCATCCCGTATCAGTGGATCTTCCCTCCGGAGGAACAGATTTTCGGCACCAGCGTGATTGTGCCGCGTCATGTGCGCCGCGCCGGTCTCAATACCATCGACCCGACCATCAAGAACTTTCAGTGGGGTGACCTGTCCGCCGCCATCCGTGAGGCCCATGATCGCGGCGCACGTAGCGCAGTTCTGCTGGATGCCGATAATTGCGTGGCCGAGGGTCCGGGCTTTAATGTGGTTCTGGTGAAGGACGGCGCACTGGTGAGTCCGAGTCGTAACGCACTGCCGGGCATCACCCGCAAAACCGTGTACGAAATCGCAGCAGCCAAAGGCATCGAAACCATGCTGCGCGACGTGACCAGCAGTGAACTGTATGAGGCAGATGAGCTGATGGCCGTGAGCACCGGAGGTGGTGTGACCCCTATTACCAGCCTGGATGGCGAGCAGGTTGGCAACGGTGAGCCGGGCCCGATTACCGTGGCCATTCGTGATCGCTTTTGGGCACTGATGGATGAGCCGAGCAGCCTGATTGAGGCCATTGACTATTAA)可以催化目标底物得到产物，但是催化活性较差。
为了扩大酶的底物谱、提高酶对大位阻底物的催化能力，增强其稳定性，本申请以pET22b为表达载体，通过定点突变、饱和突变、易错PCR以及组合突变等方式在转氨酶上引入突变位点，获得含有突变基因的质粒，以BL21(DE3)为表达菌株，在IPTG的诱导下获得突变体蛋白。然后对突变体进行活性检测，挑选活性提高的突变体。
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。转氨酶诱导表达最 佳条件：25℃，0.1mM IPTG诱导过夜。然后通过超声破碎细胞的方法获得粗酶。
反应验证条件：配置底物母液(底物溶于DMSO中)和PLP母液(10mg 5-磷酸吡哆醛溶于1mL的buffer中)，在10mL反应器中依次加入硼酸buffer、PLP母液、6M异丙胺盐酸盐溶液、底物母液，最后加入酶(20％酶液)，在摇床中200rpm反应，反应结束后在反应体系中加入乙腈，震荡混匀后12000rpm，离心3min，取上清稀释适合的倍数，送HPLC检测活性。
前期通过定点突变和单点饱和突变的理性方式对转氨酶进行初步的改造，用底物1进行活力验证：反应式如下，结果如表1所示。
Figure PCTCN2022093313-appb-000004
表1
突变体 酶活 反应条件
Template + A
T70A + A
Y75F ++ A
Y75A - A
G77V - A
G77A + A
Y144F + A
Y144A + A
M130Y - A
M130A - A
Q163L + A
Q163A ++ A
I165A ++ A
W164F + A
W164A + A
W200F N.D. A
W200A - A
G232A - A
G232V - A
F233Y + A
S290A - A
S290T + A
T291A ++ A
G292A ++ A
G292V - A
T291A+S290L + A
T291A+S290V ++ A
T291A+S290T ++ A
T291Q ++ A
T291E - A
T291V + A
T291F - A
T291T + A
T291I + A
T291K + A
T291C ++ A
T291Y - A
T291G + A
T291N + A
T291L + A
T291S ++ A
T291W N.D. A
T291R - A
T291H - A
T291P - A
T291M ++ A
T291A+G292A +++ A
T291A+G292L + A
T291A+G292I + A
T291A+G292D ++ A
T291A+G292A+Y75T ++ A
T291A+G292A+G77E +++ A
T291A+G292A+M130I +++ A
T291A+G292A+M130L +++ A
T291A+G292A+Y144S ++ A
T291A+G292A+Y144G ++ A
T291A+G292A+Y144I +++ A
T291A+G292A+Y144V +++ A
T291A+G292A+Y144P +++ A
T291A+G292A+Y144L +++ A
T291A+G292A+Y144H ++ A
T291A+G292A+Q163S +++ A
T291A+G292A+Q163T +++ A
T291A+G292A+Q163L ++ A
T291A+G292A+Q163N +++ A
T291A+G292A+Q163V ++ A
T291A+G292A+W164F +++ A
T291A+G292A+W164R ++ A
T291A+G292A+W164L ++ A
T291A+G292A+W164T ++ A
T291A+G292A+I165A ++ A
T291A+G292A+I165V +++ A
T291A+G292A+I165L ++ A
T291A+G292A+W200G ++ A
T291A+G292A+W200H - A
T291A+G292A+L232F +++ A
T291A+G292A+F233W ++ A
T291A+G292A+F233Y +++ A
T291A+G292A+T70G +++ A
T291A+G292A+T70M +++ A
T291A+G292A+T70A +++ A
T291A+G292A+T70S ++ A
T291A+G292A+T70C ++++ A
T291A+G292A+W164Y ++ A
备注：
1)反应条件A：5wt酶、PLP 2mg、DMSO 20％、IPN 30eq、500V、0.1M Tris-Cl9.0、37℃、40h；
2)N.D.表示未检测到产品生成，-表示0～1％，+表示1～5％，++表示5～10％，+++表示10～15％，++++表示＞15％。
上述结果说明，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催化活力和稳定性，通过饱和突变以及组合突变的方式和定向筛选的方法对突变体的耐受性进行改造。效果验证反应式如下，结果如表2所示。
Figure PCTCN2022093313-appb-000005
表2
突变体 酶活 反应条件
T291A+G292A+T70C + B
T291A+G292A+T70C+W164F ++ B
T291A+G292A+T70C+F233Y ++ B
T291A+G292A+T70C+I165V +++ B
T291A+G292A+T70C+Y144M+G145N +++ B
T291A+G292A+T70C+Y144M+G145S +++ B
T291A+G292A+T70C+G145L ++ B
T291A+G292A+T70C+Y69L ++ B
T291A+G292A+T70C+T142G ++ B
T291A+G292A+T70C+I160G +++++ B
T291A+G292A+T70C+I160T +++ B
T291A+G292A+T70C+I160S +++ B
T291A+G292A+T70C+I160V +++ B
T291A+G292A+T70C+I160C ++ B
T291A+G292A+T70C+S204L ++++ B
T291A+G292A+T70C+S204M ++++ B
T291A+G292A+T70C+V244G +++ B
T291A+G292A+T70C+V244R +++ B
T291A+G292A+T70C+V244Y +++ B
T291A+G292A+T70C+V244S ++++ B
T291A+G292A+T70C+V244K +++ B
T291A+G292A+T70C+V244M ++++ B
T291A+G292A+T70C+I160G+H151A +++++ B
T291A+G292A+T70C+I160G+H151D +++++ B
T291A+G292A+T70C+I160G+H151Y +++++ B
T291A+G292A+T70C+I160G+H151Q +++++ B
T291A+G292A+T70C+I160G ++ C
T291A+G292A+T70C+I160G+S204L +++ C
T291A+G292A+T70C+I160G+V244S +++ C
T291A+G292A+T70C+I160G+S204L+V244S +++ C
T291A+G292A+T70C+I160G+T134I ++ C
T291A+G292A+T70C+I160G+A242S ++ C
T291A+G292A+T70C+I160G+A242Q ++ C
T291A+G292A+T70C+I160G+T270G ++ C
T291A+G292A+T70C+I160G+T270H ++ C
T291A+G292A+T70C+I160G+S278L ++ C
T291A+G292A+T70C+I160G+S278R +++ C
T291A+G292A+T70C+I160G+S278K ++ C
T291A+G292A+T70C+I160G+E282C + C
T291A+G292A+T70C+1160G+E282R ++ C
T291A+G292A+T70C+I160G+E282K + C
T291A+G292A+T70C+I160G+M326Q ++ C
T291A+G292A+T70C+I160G+V17S ++ C
T291A+G292A+T70C+I160G+A56S ++ C
T291A+G292A+T70C+I160G+F64L ++ C
T291A+G292A+T70C+I160G+P167A ++ C
T291A+G292A+T70C+I160G+P167K ++ C
T291A+G292A+T70C+I160G+R216E +++ C
T291A+G292A+T70C+I160G+R216K ++ C
T291A+G292A+T70C+I160G+S204L+V244S+T134I ++++ C
T291A+G292A+T70C+I160G+S204L+V244S+M326Q +++ C
T291A+G292A+T70C+I160G+S204L+V244S+T134I ++++ D
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q ++++ D
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q +++ E
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158I +++++ E
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V +++++ E
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216N +++ E
T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216K +++ E
备注：
1)反应条件B：2wt酶、PLP 4mg、DMSO 30％、IPN 30eq、200V、0.1M硼酸10.0、50℃、16h；
2)反应条件C：0.5wt酶、PLP 4g/L、DMSO 30％、IPN 30eq、100V、0.1M硼酸10.0、50℃、16h；
3)反应条件D：0.5wt酶、PLP 1g/L、DMSO 30％、IPN 30eq、100V、0.1M硼酸10.5、55℃、16h；
4)反应条件E：0.1wt酶、PLP 1g/L、DMSO 50％、IPN 5eq、100V、0.2M硼酸10.5、60℃、16h；
5)N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示>90％。
经过上述耐受性改造后的突变体经过底物1的酶活验证，突变体耐高温的稳定性得到显著的提升，Template最适反应温度为37℃，突变体T291A+G292A+T70C、突变体T291A+G292A+T70C+I160G等突变体的最适反应温度提升到50℃，突变体T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V最适反应温度提升到60℃；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20％提升到50％；而且酶活力也得到不断的逐步提高，突变体T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V的酶活力相较Template大幅度提高。为了改善突变体的立体选择性，通过饱和突变、组合突变以及易错PCR的进化方式和定向筛选的方法对突变体的选择性进行改造。效果验证反应式如下，结果如表3所示。
Figure PCTCN2022093313-appb-000006
表3
Figure PCTCN2022093313-appb-000007
Figure PCTCN2022093313-appb-000008
Figure PCTCN2022093313-appb-000009
Figure PCTCN2022093313-appb-000010
Figure PCTCN2022093313-appb-000011
“-12aa”表示去掉蛋白前端的12个氨基酸。
备注：
Figure PCTCN2022093313-appb-000012
1)转氨酶突变体ee值，ee值＝((CP1+CP)-(CP2+CP3))/(CP+CP1+CP2+CP3)
2)反应条件D：0.5wt酶、PLP 1g/L、DMSO 30％、IPN(异丙胺盐酸盐)30eq、100V、0.1M硼酸10.5、55℃、16h；
3)反应条件F：7wt酶、PLP 1g/L、DMSO 20％、IPN 30eq、500V、0.2M硼酸10.5、60℃、16h；
4)反应条件G：0.1wt酶、PLP 1g/L、DMSO 30％、IPN 10eq、200V、0.2M硼酸10.5、60℃、16h；
5)反应条件H：0.3wt酶、PLP 1g/L、DMSO 30％、IPN 10eq、100V、0.2M硼酸10.5、60℃、16h；
6)反应条件L：0.3wt酶、PLP 1g/L、DMSO 40％、IPN 10eq、100V、0.2M硼酸10.5、60℃、16h；
7)反应条件M：0.3wt酶、PLP 1g/L、DMSO 40％、IPN 10eq、100V、0.2M硼酸10.5、55℃、16h；
8)N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示>90％；
9)N.D.表示0，*表示0～10％，**表示10～50％，***表示50～80％，****表示80～90％，*****表示90～95％，******表示>95％。
上述选择性改造后的突变体经过底物1的酶活验证，突变体的选择性在不断逐步改善，突变体T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292S转氨酶突变体ee值提高至70％以上，突变体T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144W-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa转氨酶突变体ee值提高至80％以上，T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa转氨酶突变体ee值提高至90％以上，T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+T70G+G160T+T106S-12aa转氨酶突变体ee值提高至95％以上。
通过采用软件对转氨酶(Template)的三维结构进行计算机模拟分析，突变的位点大部分位于活性中心附近(活性中心位点主要包括T70、Y144、A292、Y69、I160等位点)，突变后有可能增强了底物和酶的结合，从而提高了选择性和催化效率。
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，所述突变至少包括：T291A；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，优选具有85％的同源性，更优选具有90％的同源性，进一步优选具有95％的同源性，再优选具有99％的同源性。优选的，突变包括T291A和至少如下突变位点之一：V17S、A56S、A56P、F64L、Y69L、Y69M、Y69C、Y69G、T70A、T70G、T70M、T70C、T70S、Y75F、G77E、G77S、G77V、G77T、G77L、I78H、T106S、M130I、M130L、N132W、N132A、T134I、T134A、T134V、Y139S、G140C、S141G、K142G、K142V、Y144I、Y144V、Y144P、Y144L、Y144M、Y144F、E145N、E145S、E145L、E145H、E145D、A148T、A148Q、H151A、H151D、H151Y、H151Q、R152K、R152T、Y158I、Y158V、I160G、I160T、I160S、I160V、I160C、I160T、Q163S、Q163T、Q163N、Q163A、W164F、I165L、I165H、I165A、I165V、P167A、P167K、Q171T、W200A、S204L、S204M、R216E、R216K、R216N、G232F、F233Y、A242S、A242Q、V244G、V244R、V244Y、V244S、V244K、V244M、T270G、T270H、S278L、S278R、S278K、E282C、E282R、E282K、T291A、T291Q、T291C、T291S、T291M、T291T、G292A、G292S和M326Q。
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：T291A+S290L、T291A+S290V、T291A+S290T、T291A+G292A、T291A+G292L、T291A+G292I、T291A+G292D、T291A+G292A+Y75T、T291A+G292A+G77E、T291A+G292A+M130I、T291A+G292A+M130L、T291A+G292A+Y144S、T291A+G292A+Y144G、T291A+G292A+Y144I、T291A+G292A+Y144V、T291A+G292A+Y144P、T291A+G292A+Y144L、T291A+G292A+Y144H、T291A+G292A+Q163S、T291A+G292A+Q163T、T291A+G292A+Q163L、T291A+G292A+Q163N、T291A+G292A+Q163V、T291A+G292A+W164F、T291A+G292A+W164R、T291A+G292A+W164L、T291A+G292A+W164T、T291A+G292A+I165A、T291A+G292A+I165V、T291A+G292A+I165L、T291A+G292A+W200G、T291A+G292A+T70C、T291A+G292A+T70C+W164F、T291A+G292A+T70C+F233Y、T291A+G292A+T70C+I165V、T291A+G292A+T70C+Y144M+G145N、T291A+G292A+T70C+Y144M+G145S、T291A+G292A+T70C+G145L、T291A+G292A+T70C+Y69L、T291A+G292A+T70C+T142G、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160T、T291A+G292A+T70C+I160S、T291A+G292A+T70C+I160V、T291A+G292A+T70C+I160C、T291A+G292A+T70C+S204L、T291A+G292A+T70C+S204M、T291A+G292A+T70C+V244G、T291A+G292A+T70C+V244R、T291A+G292A+T70C+V244Y、T291A+G292A+T70C+V244S、T291A+G292A+T70C+V244K、T291A+G292A+T70C+V244M、T291A+G292A+T70C+I160G+H151A、T291A+G292A+T70C+I160G+H151D、T291A+G292A+T70C+I160G+H151Y、T291A+G292A+T70C+I160G+H151Q、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160G+S204L、T291A+G292A+T70C+I160G+V244S、T291A+G292A+T70C+I160G+S204L+V244S、T291A+G292A+T70C+I160G+T134I、T291A+G292A+T70C+I160G+A242S、T291A+G292A+T70C+I160G+A242Q、T291A+G292A+T70C+I160G+T270G、T291A+G292A+T70C+I160G+T270H、T291A+G292A+T70C+I160G+S278L、T291A+G292A+T70C+I160G+S278R、T291A+G292A+T70C+I160G+S278K、T291A+G292A+T70C+I160G+E282C、T291A+G292A+T70C+I160G+E282R、T291A+G292A+T70C+I160G+E282K、T291A+G292A+T70C+I160G+M326Q、T291A+G292A+T70C+I160G+V17S、T291A+G292A+T70C+I160G+A56S、T291A+G292A+T70C+I160G+F64L、T291A+G292A+T70C+I160G+P167A、T291A+G292A+T70C+I160G+P167K、T291A+G292A+T70C+I160G+R216E、T291A+G292A+T70C+I160G+R216K、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216N、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216K、T291A+G292A+L232F、 T291A+G292A+F233W、T291A+G292A+F233Y、T291A+G292A+T70G、T291A+G292A+T70M、T291A+G292A+T70A、T291A+G292A+T70S、T291A+G292A+T70C、T291A+G292A+W164Y、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+G77V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+N132A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77S、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+Y144F、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144W-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144P-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+N132R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+I78V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A291I-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+I78H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+T142V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145D-12aa、 T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+F233Y-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145D-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69G-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+S141G-12aa、T291A+G292A+T70A+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+Y139S-12aa、 T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+S292A-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+A291T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+I134V-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+T106S-12aa、或T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+Q171T-12aa。
根据本发明一种典型的实施方式，提供一种DNA分子。该DNA分子编码上述耐有机溶剂的转氨酶突变体。该DNA分子编码的上述转氨酶突变体具有很好的有机溶剂耐受性和高pH耐受性，并且具有高可溶性表达特性以及高活力特性。
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、pKK232-8、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，真核细胞为酵母。
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耐有机溶剂的转氨酶突变体。由于本发明的上述转氨酶突变体具有扩大的酶底物谱、对大位阻底物的催化能力较高；同时稳定性增强，可在高浓度溶剂和高温度条件下进行催化反应，进而此突变体的应用可以提高反应速率，提高酶稳定性，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。
进一步地，酮类化合物为
Figure PCTCN2022093313-appb-000013
其中，R 1为H、未被取代或被卤素任选取代的C1～C10烷基、未被取代或被卤素任选取代C5～C10环烷基、未被取代或被卤素任选取代的C5～C10芳基或C5～C10杂芳基，R 2为H、卤素、未被取代或被卤素、NH 3或带Boc、Cbz保护的NH 3中至少一个基团所任选取代C1～C10烷基，R 3为未被取代或被卤素任选取代的C6-12的芳基、未被取代或被卤素任选取代的C3-6的杂环基；
优选的，所述酮类化合物为
Figure PCTCN2022093313-appb-000014
(底物1)、
Figure PCTCN2022093313-appb-000015
(底物2)、
Figure PCTCN2022093313-appb-000016
(底物3)、
Figure PCTCN2022093313-appb-000017
(底物4)或
Figure PCTCN2022093313-appb-000018
(底物5)。
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7～10.5，优选为8～10，更优选为9～10，也就是说pH的取值可以任选为7～10.5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。
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。
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
Template及突变体催化酮化合物生成手性胺，在反应条件M的情况下验活，结果可见Template对底物催化活力很差，而Template突变体具有较好的催化活性，并且突变体T291A+G292A+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+T70G+G160T+T106S-12aa转氨酶突变体ee值提高至90％以上。经过本发明的改造后，Template突变体的获得了对空间位阻较大底物的催化活力，部分突变体活力极大提高，而且突变体的立体选择性也大幅度提高，扩大了底物谱。反应式如下，结果如表4所示。
Figure PCTCN2022093313-appb-000019
表4
Figure PCTCN2022093313-appb-000020
备注：
1)反应条件M：0.3wt酶、PLP 1g/L、DMSO 40％、IPN 10eq、100V、0.2M硼酸pH＝10.5、55℃、16h；
2)N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示>90％。
3)N.D.表示0，*表示0～10％，**表示10～50％，***表示50～80％，****表示80～90％，*****表示90～95％，******表示>95％。
实施例2
Template及突变体催化酮化合物生成手性胺，在反应条件N的情况下验活，结果可见Template对底物无催化活力，而Template突变体具有较好的催化活性。经过本发明的改造后，Template突变体扩大了催化底物谱。反应式如下，结果如表5所示。
Figure PCTCN2022093313-appb-000021
表5
Figure PCTCN2022093313-appb-000022
备注：
反应条件M：1wt酶、PLP 1g/L、DMSO 20％、IPN 10eq、100V、0.2M硼酸pH＝10.5、50℃、16h；
N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示90％～95％，++++++表示>95％。
实施例3
Template及突变体催化酮化合物生成手性胺，在反应条件N的情况下验活，结果可见 Template对底物无催化活力，而Template突变体具有较好的催化活性。经过本发明的改造后，Template突变体扩大了催化底物谱。反应式如下，结果如表6所示。
Figure PCTCN2022093313-appb-000023
表6
Figure PCTCN2022093313-appb-000024
备注：
反应条件M：1wt酶、PLP 1g/L、DMSO 20％、IPN 10eq、100V、0.2M硼酸pH＝10.5、50℃、16h；
N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示90％～95％，++++++表示>95％。
实施例4
Template及突变体催化酮化合物生成手性胺，在反应条件N的情况下验活，结果可见Template对底物无催化活力，而Template突变体具有较好的催化活性。经过本发明的改造后，Template突变体扩大了催化底物谱。反应式如下，结果如表7所示。
Figure PCTCN2022093313-appb-000025
表7
Figure PCTCN2022093313-appb-000026
Figure PCTCN2022093313-appb-000027
备注：
反应条件M：1wt酶、PLP 1g/L、DMSO 20％、IPN 10eq、100V、0.2M硼酸pH＝10.5、50℃、16h；
2)N.D.表示未检测到产品生成，-表示0～20％，+表示20～40％，++表示40～60％，+++表示60～80％，++++表示80～90％，+++++表示90～100％。
实施例5
Template及突变体催化酮化合物生成手性胺，在反应条件N的情况下验活，结果可见Template对底物无催化活力，而Template突变体具有较好的催化活性。经过本发明的改造后，Template突变体扩大了催化底物谱。反应式如下，结果如表8所示。
Figure PCTCN2022093313-appb-000028
表8
Figure PCTCN2022093313-appb-000029
备注：
反应条件M：1wt酶、PLP 1g/L、DMSO 20％、IPN 10eq、100V、0.2M硼酸pH＝10.5、50℃、16h；
2)N.D.表示未检测到产品生成，-表示0～10％，+表示10～20％，++表示20～50％，+++表示50～80％，++++表示80～90％，+++++表示>90％。
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：本发明的突变体扩大了酶的底物谱、提高了酶对大位阻底物的催化能力；同时增强了其稳定性，可在高浓度溶剂和高温度条件下进行催化反应，进而此突变体的应用可以提高反应速率，提高酶稳定性，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。
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 (21)

  1. 一种转氨酶突变体，其特征在于，所述转氨酶突变体是由SEQ ID NO：1所示的氨基酸序列发生突变得到，所述突变至少包括：T291A；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，优选具有85％的同源性，更优选具有90％的同源性。
  2. 根据权利要求1所述的转氨酶突变体，其特征在于，所述突变包括T291A和至少如下突变位点之一：V17S、A56S、A56P、F64L、Y69L、Y69M、Y69C、Y69G、T70A、T70G、T70M、T70C、T70S、Y75F、G77E、G77S、G77V、G77T、G77L、I78H、T106S、M130I、M130L、N132W、N132A、T134I、T134A、T134V、Y139S、G140C、S141G、K142G、K142V、Y144I、Y144V、Y144P、Y144L、Y144M、Y144F、E145N、E145S、E145L、E145H、E145D、A148T、A148Q、H151A、H151D、H151Y、H151Q、R152K、R152T、Y158I、Y158V、I160G、I160T、I160S、I160V、I160C、I160T、Q163S、Q163T、Q163N、Q163A、W164F、I165L、I165H、I165A、I165V、P167A、P167K、Q171T、W200A、S204L、S204M、R216E、R216K、R216N、G232F、F233Y、A242S、A242Q、V244G、V244R、V244Y、V244S、V244K、V244M、T270G、T270H、S278L、S278R、S278K、E282C、E282R、E282K、T291A、T291Q、T291C、T291S、T291M、T291T、G292A、G292S和M326Q。
  3. 根据权利要求2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：T291A+S290L、T291A+S290V、T291A+S290T、T291A+G292A、T291A+G292L、T291A+G292I、T291A+G292D、T291A+G292A+Y75T、T291A+G292A+G77E、T291A+G292A+M130I、T291A+G292A+M130L、T291A+G292A+Y144S、T291A+G292A+Y144G、T291A+G292A+Y144I、T291A+G292A+Y144V、T291A+G292A+Y144P、T291A+G292A+Y144L、T291A+G292A+Y144H、T291A+G292A+Q163S、T291A+G292A+Q163T、T291A+G292A+Q163L、T291A+G292A+Q163N、T291A+G292A+Q163V、T291A+G292A+W164F、T291A+G292A+W164R、T291A+G292A+W164L、T291A+G292A+W164T、T291A+G292A+I165A、T291A+G292A+I165V、T291A+G292A+I165L、T291A+G292A+W200G、T291A+G292A+T70C、T291A+G292A+T70C+W164F、T291A+G292A+T70C+F233Y、T291A+G292A+T70C+I165V、T291A+G292A+T70C+Y144M+G145N、T291A+G292A+T70C+Y144M+G145S、T291A+G292A+T70C+G145L、T291A+G292A+T70C+Y69L、T291A+G292A+T70C+T142G、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160T、T291A+G292A+T70C+I160S、T291A+G292A+T70C+I160V、T291A+G292A+T70C+I160C、T291A+G292A+T70C+S204L、T291A+G292A+T70C+S204M、T291A+G292A+T70C+V244G、T291A+G292A+T70C+V244R、T291A+G292A+T70C+V244Y、T291A+G292A+T70C+V244S、T291A+G292A+T70C+V244K、 T291A+G292A+T70C+V244M、T291A+G292A+T70C+I160G+H151A、T291A+G292A+T70C+I160G+H151D、T291A+G292A+T70C+I160G+H151Y、T291A+G292A+T70C+I160G+H151Q、T291A+G292A+T70C+I160G、T291A+G292A+T70C+I160G+S204L、T291A+G292A+T70C+I160G+V244S、T291A+G292A+T70C+I160G+S204L+V244S、T291A+G292A+T70C+I160G+T134I、T291A+G292A+T70C+I160G+A242S、T291A+G292A+T70C+I160G+A242Q、T291A+G292A+T70C+I160G+T270G、T291A+G292A+T70C+I160G+T270H、T291A+G292A+T70C+I160G+S278L、T291A+G292A+T70C+I160G+S278R、T291A+G292A+T70C+I160G+S278K、T291A+G292A+T70C+I160G+E282C、T291A+G292A+T70C+I160G+E282R、T291A+G292A+T70C+I160G+E282K、T291A+G292A+T70C+I160G+M326Q、T291A+G292A+T70C+I160G+V17S、T291A+G292A+T70C+I160G+A56S、T291A+G292A+T70C+I160G+F64L、T291A+G292A+T70C+I160G+P167A、T291A+G292A+T70C+I160G+P167K、T291A+G292A+T70C+I160G+R216E、T291A+G292A+T70C+I160G+R216K、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158I、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y158V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216N、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+R216K、T291A+G292A+L232F、T291A+G292A+F233W、T291A+G292A+F233Y、T291A+G292A+T70G、T291A+G292A+T70M、T291A+G292A+T70A、T291A+G292A+T70S、T291A+G292A+T70C、T291A+G292A+W164Y、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+G77V、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77L、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+N132A、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77S、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+W200A+A56P+Y144F、 T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+A292G+G77T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144W-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+L144P-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+N132R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144L+G77T+I165H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+I78V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A291I-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+I78H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+T142V-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+G145D-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+R152T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+F233Y-12aa、 T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+G145D-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148T-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+A148Q-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M+R152K-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69M-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69G-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70S+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70C+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+S141G-12aa、T291A+G292A+T70A+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G140C-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+Y139S-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+S292A-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+A291T-12aa、T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+I134V-12aa、 T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+T106S-12aa、或T291A+G292A+T70G+I160G+S204L+V244S+T134I+M326Q+Y144W+G77T+I165L+A292S+S278R+Y69H+G160T+Q171T-12aa。
  4. 一种DNA分子，其特征在于，所述DNA分子编码权利要求1至3中任一项所述的转氨酶突变体。
  5. 一种重组质粒，其特征在于，所述重组质粒含有权利要求4所述的DNA分子。
  6. 根据权利要求5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、pKK232-8、pUC-18或pUC-19。
  7. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求5或6所述的重组质粒。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述宿主细胞包括原核细胞或真核细胞。
  9. 根据权利要求8所述的宿主细胞，其特征在于，所述原核细胞为大肠杆菌BL21-DE3细胞或大肠杆菌Rosetta-DE3细胞；所述真核细胞为酵母。
  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3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 PCTCN2022093313-appb-100001
    其中，R 1为H、未被取代或被卤素任选取代的C1～C10烷基、未被取代或被卤素任选取代C5～C10环烷基、未被取代或被卤素任选取代的C5～C10芳基或C5～C10杂芳基，R 2为H、卤素、未被取代或被卤素、NH 3或带Boc、Cbz保护的NH 3中至少一个基团所任选取代C1～C10烷基，R 3为未被取代或被卤素任选取代的C6-12的芳基、未被取代或被卤素任选取代的C3-6的杂环基。
  12. 根据权利要求11所述的方法，其特征在于，所述酮类化合物为
    Figure PCTCN2022093313-appb-100002
    Figure PCTCN2022093313-appb-100003
  13. 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。
  14. 根据权利要求13所述的方法，其特征在于，所述氨基供体为异丙胺。
  15. 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。
  16. 根据权利要求15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8～10.5。
  17. 根据权利要求16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10.5。
  18. 根据权利要求15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的温度为25～60℃。
  19. 根据权利要求18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的温度为30～55℃。
  20. 根据权利要求19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的温度为50℃。
  21. 根据权利要求15所述的方法，其特征在于，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％。
PCT/CN2022/093313 2022-04-21 2022-05-17 转氨酶突变体及其应用 WO2023201809A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210426193.1A CN114525265B (zh) 2022-04-21 2022-04-21 转氨酶突变体及其应用
CN202210426193.1 2022-04-21

Publications (1)

Publication Number Publication Date
WO2023201809A1 true WO2023201809A1 (zh) 2023-10-26

Family

ID=81627777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/093313 WO2023201809A1 (zh) 2022-04-21 2022-05-17 转氨酶突变体及其应用

Country Status (2)

Country Link
CN (2) CN115851649A (zh)
WO (1) WO2023201809A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628742A (zh) * 2019-10-25 2019-12-31 凯莱英医药化学（阜新）技术有限公司 转氨酶突变体及其应用
CN111032682A (zh) * 2017-06-14 2020-04-17 科德克希思公司 用于工业生物催化的工程化转氨酶多肽
US20210317421A1 (en) * 2020-04-10 2021-10-14 Codexis, Inc. Engineered transaminase polypeptides

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105018440B (zh) * 2014-04-24 2018-06-05 上海弈柯莱生物医药科技有限公司 一种转氨酶及其在合成西他列汀中间体中的应用
CN107828751B (zh) * 2017-11-06 2021-02-26 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及其应用
CN108048419B (zh) * 2017-11-15 2020-10-23 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及其应用
CN108913671B (zh) * 2018-06-15 2020-07-24 浙江科技学院 一种ω-转氨酶突变体及其应用
US20220380817A1 (en) * 2019-10-25 2022-12-01 Asymchem Laboratories (Fuxin) Co., Ltd. Transaminase mutant and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111032682A (zh) * 2017-06-14 2020-04-17 科德克希思公司 用于工业生物催化的工程化转氨酶多肽
CN110628742A (zh) * 2019-10-25 2019-12-31 凯莱英医药化学（阜新）技术有限公司 转氨酶突变体及其应用
US20210317421A1 (en) * 2020-04-10 2021-10-14 Codexis, Inc. Engineered transaminase polypeptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN ANONYMOUS : "aminotransferase class IV [Sciscionella sp. SE31]", XP093103502, retrieved from NCBI *
WANG CHEN, TANG KEXIN, DAI YA, JIA HONGHUA, LI YAN, GAO ZHEN, WU BIN: "Identification, Characterization, and Site-Specific Mutagenesis of a Thermostable ω-Transaminase from Chloroflexi bacterium", ACS OMEGA, ACS PUBLICATIONS, US, vol. 6, no. 26, 6 July 2021 (2021-07-06), US , pages 17058 - 17070, XP093103505, ISSN: 2470-1343, DOI: 10.1021/acsomega.1c02164 *
XU WEI, LI YAN, YAN MING: "The Applications of Site-directed Mutagenesis in Aminotransferase", JOURNAL OF YANCHENG INSTITUTE OF TECHNOLOGY (NATURAL SCIENCE EDITION), vol. 17, no. 4, 31 December 2004 (2004-12-31), pages 55 - 58, XP093103510, ISSN: 1671-5322, DOI: 10.16018/j.cnki.32-1650/n.2004.04.015 *

Also Published As

Publication number Publication date
CN115851649A (zh) 2023-03-28
CN114525265B (zh) 2022-09-30
CN114525265A (zh) 2022-05-24

Similar Documents

Publication Publication Date Title
CN107828751B (zh) 转氨酶突变体及其应用
CN108048417B (zh) 酮还原酶突变体及其应用
CN110628742B (zh) 转氨酶突变体及其应用
JP7022819B2 (ja) トランスアミナーゼ突然変異体及びその使用
CN113736759B (zh) 转氨酶突变体及其应用
WO2019153183A1 (zh) 一种单加氧酶突变体及其制备方法和应用
JP7462795B2 (ja) トランスアミナーゼ突然変異体及びその使用
WO2022160408A1 (zh) 酯酶突变体及其应用
JP6988000B2 (ja) ケトレダクターゼ変異体及びその応用
WO2021077425A1 (zh) 转氨酶突变体及其应用
WO2021081713A1 (zh) 转氨酶突变体及其应用
JP7083408B2 (ja) アミノ酸デヒドロゲナーゼ突然変異体及びその応用
JP7263557B2 (ja) アミノ基転移酵素突然変異体及びその応用
JP7498244B2 (ja) モノオキシゲナーゼ突然変異体及びその使用
WO2023201809A1 (zh) 转氨酶突变体及其应用
JP7293351B2 (ja) モノオキシゲナーゼ突然変異体及びその応用
CN114277011B (zh) 转氨酶突变体及其应用
CN116814574A (zh) 一种亚胺还原酶突变体、重组基因工程菌及其在(r)-2-甲基吡咯烷合成中的应用
JPH05219978A (ja) 核酸関連物質の酵素的製造法及びそれに使用する酵素調製物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22938043

Country of ref document: EP

Kind code of ref document: A1