WO2024113508A1 - 转氨酶突变体及应用 - Google Patents

转氨酶突变体及应用 Download PDF

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WO2024113508A1
WO2024113508A1 PCT/CN2023/077455 CN2023077455W WO2024113508A1 WO 2024113508 A1 WO2024113508 A1 WO 2024113508A1 CN 2023077455 W CN2023077455 W CN 2023077455W WO 2024113508 A1 WO2024113508 A1 WO 2024113508A1
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group
substituted
alkylene
alkyl
amino acid
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PCT/CN2023/077455
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French (fr)
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洪浩
詹姆斯•盖吉
张娜
焦学成
马玉磊
牟慧艳
马天娇
赵桐
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凯莱英生命科学技术(天津)有限公司
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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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)

Definitions

  • the present invention relates to the field of enzyme catalysis, and in particular to a transaminase mutant and application thereof.
  • Chiral amines are the structural units of many important biologically active molecules and key intermediates for the synthesis of many chiral drugs.
  • the chemical method has the disadvantages of long reaction routes and harsh conditions, the use of toxic transition metal catalysts in the synthesis, low product stereoselectivity and low yield; and the theoretical maximum yield of the biological resolution method is only 50%. Both methods have certain limitations in scale-up production.
  • transaminase The asymmetric synthesis of chiral amines catalyzed by transaminase has attracted more and more attention due to its advantages such as high selectivity, high conversion rate, mild reaction conditions and environmental friendliness, and has now become a widely used method for preparing chiral amines.
  • most wild-type transaminases have a limited range of substrates and are generally difficult to synthesize large sterically hindered chiral amine compounds (referring to the substituents on one side next to the carbonyl group of the catalytic substrate being larger than the methyl group).
  • CN114875006A discloses a transaminase mutant that can efficiently synthesize chiral amines, especially large sterically hindered chiral amines.
  • these mutants have relatively poor stability when used in industrial applications and are difficult to adapt to extreme environments such as high temperature and high concentration of organic solvents in the amplification process.
  • transaminase in order to further adapt to the industrial production requirements of synthesizing large sterically hindered chiral amines, we can improve the stability of transaminase under extreme conditions through enzyme evolution, so as to further promote the immobilization and continuous application of transaminase, improve production efficiency, reduce industrial production costs, and reduce industrial waste emissions.
  • the main purpose of the present invention is to provide a transaminase mutant and its application, so as to solve the problem in the prior art that the transaminase has poor activity or tolerance in catalyzing the industrial production of large sterically hindered chiral amine compounds.
  • a transaminase mutant comprising (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein having amino acid mutations at at least one of the following sites of the amino acid sequence in (a): V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107 or L449, and having transaminase activity; and (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having transaminase function.
  • amino acid mutations in (b) are each independently selected from the following: V315T or V315R or V315D or V315A or V315K or V315H; I91Q or I91M or I91N or I91G or I91F or I91D; V124C or V124S or V124Y or V124P or V124M or V124A or V124I; Y116F or Y116Y or Y116A or Y116R or Y116S or Y116G or Y116H or Y116L; A286I or A286R or A286Q or A286F or A286D or A286M or A286I; C418L or C418W or C418R or C418D or C418F or C418A; T87A or T87Y or T87N or T87V or T87F or T87E; S301A or S301G or S301H or S301 K or S301W or S301I or S
  • the mutation of the transaminase mutant includes any of the following amino acid mutations: V315T; V315R; V315D; V315A; V315K; V315H; V315T+I91Q; V315T+I91M; V315T+I91N; V315T+I91G; V315T+I91F; V315T+I91D; V315T+V124C; V315T+V124S; V315T+V124Y; V315T+V124P; V315T+V124M; V315T+V124A; V315T+V124C+I91N; V315T+V124C+I91Q; V315T+V124C+I91M; V315T+ V124C+I91G; V315T+V124C+I91F; V315T+I91Q+V124S; V315T+I91Q+V124I; V315T
  • a DNA molecule which encodes the above transaminase mutant.
  • a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above DNA molecule.
  • a host cell is provided, wherein the host cell is transformed with the above recombinant plasmid.
  • a method for preparing a chiral amine compound comprising using the above transaminase mutant to perform a transamination reaction on a ketone substrate shown in formula I under the action of an amino donor to obtain a chiral amine compound,
  • Ar1 is selected from the first substituted aryl, the first unsubstituted aryl, the substituted arylene or unsubstituted arylene, the substituted heteroarylene or unsubstituted heteroarylene;
  • Ar2 is selected from the second substituted aryl, the second unsubstituted aryl, the substituted cycloalkyl or unsubstituted cycloalkyl, the alkyl or alkylene;
  • R is selected from H, alkyl, alkylene or alkylidene, the number of C atoms in the alkyl, alkylene or alkylidene is selected from 1 to 5, and the alkyl, alkylene or alkylidene includes substituted alkyl, alkylene or alkylidene or unsubstituted alkyl, alkylene or alkylidene; when R is selected from alkyl, alkylene or alkylidene, the alkyl, alkylene or alkylidene is
  • substituents of the first substituted aryl group, the second substituted aryl group or the substituted arylene group are each independently selected from halogen.
  • substituents are each independently located at any one or more positions of the ortho position, meta position or para position of the first substituted aryl group, the second substituted aryl group or the substituted arylene group.
  • halogen is selected from F, Cl or Br.
  • Ar1 is selected from unsubstituted heteroarylene
  • Ar2 is selected from a second substituted aryl
  • the substituent of the second substituted aryl is selected from halogen
  • R is selected from a substituted alkylene
  • the number of C atoms of the substituted alkylene is selected from 1 to 5
  • the substituent is selected from hydroxyl
  • the substituted alkylene is connected to Ar1 to form a ring.
  • Ar1 is selected from a first substituted aromatic group or a first unsubstituted aromatic group, the substituent of the first substituted aromatic group is selected from methyl or Cl, Ar2 is selected from an unsubstituted cycloalkyl group, an alkyl group or a second unsubstituted aromatic group, the number of C atoms of the unsubstituted cycloalkyl group is selected from 3 to 5, the alkyl group is selected from isopropyl or ethyl, R is selected from an alkyl group, and the alkyl group is selected from isopropyl, methyl or ethyl.
  • Ar1 is selected from a first substituted aryl group or a first unsubstituted aryl group
  • the substituent of the first substituted aryl group is selected from methyl or -CH 2 CH 2 OH
  • Ar2 is selected from a second unsubstituted aryl group or an alkyl group
  • the alkyl group is selected from methyl or isopropyl
  • R is selected from a substituted alkylene group, the number of C atoms of the substituted alkylene group is selected from 1 to 5, the substituent is selected from hydroxyl, and the substituted alkylene group is connected to Ar1 to form a ring.
  • ketone substrate is selected from
  • transaminase mutant derived from Chromobacterium violaceum (SEQ ID NO: 1) was used as the parent, and protein engineering modifications such as saturation mutation and combinatorial mutation were carried out to obtain a transaminase mutant with greatly improved activity and tolerance in the reaction of catalyzing large sterically hindered chiral amines.
  • Large sterically hindered chiral compound in the present application, refers to a ketone compound in which the group next to the prochiral carbonyl group is larger than the methyl group, wherein the group larger than the methyl group may be ethyl, propyl, tert-butyl or tert-butyl phenyl, etc.
  • the inventors attempted to modify the transaminase by enzyme evolution, thereby improving the various properties of the transaminase under extreme conditions, so that the production efficiency can be improved in industrial production, and thus proposed a series of protection schemes of the present application.
  • a transaminase mutant comprising: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein having amino acid mutations at at least one of the following sites of the amino acid sequence in (a): V315, I91, V124, Y116, A286, C418, T87, S301, S27, V31, R34, M64, A74, R77, S101, T117, N151, L213, T285, T107 or L449, and having transaminase activity; (c) a protein having more than 80% homology with the amino acid sequence defined in any one of (a) and (b) and having transaminase function.
  • the amino acid sequence shown in SEQ ID NO: 1 is a mutant of transaminase from Chromobacterium violaceum.
  • the homology modeling of the amino acid sequence was analyzed by computer simulation, and the model structure was compared with different large sterically hindered ketone compounds.
  • Molecular docking was performed on the target protein, and 19 amino acid residues were found, including S27, V31, R34, M64, A74, R77, T87, I91, S101, Y116, T117, V124, N151, L213, T285, A286, V315, C418 and L449. These amino acid sites may affect the catalytic activity and stability of the protein.
  • proteins with transaminase function or even enhanced transaminase function can be obtained.
  • changes can be made at non-critical mutation sites and active sites, and proteins with more than 80% homology to the above amino acid sequence and transaminase function can be obtained.
  • SEQ ID NO: 1 The sequence of SEQ ID NO: 1 is as follows:
  • the amino acid mutations in (b) are each independently selected from the following: V315T or V315R or V315D or V315A or V315K or V315H; I91Q or I91M or I91N or I91G or I91F or I91D; V124C or V124S or V124Y or V124P or V124M or V124A or V124I; Y116F or Y116Y or Y116A or Y116R or Y116S or Y116G or Y116H or Y116L; A286I or A286R or A286Q or A286F or A286D or A286M or A286I; C418L or C418W or C418R or C418D or C418F or C418A; T87A or T87Y or T87N or T87V or T87F or T87E; S301A or S301G or S301H or S301K or S301W or
  • amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • Constant amino acid replacements include but are not limited to:
  • Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;
  • hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;
  • Amino acids with positively charged side chains are replaced by other amino acids with positively charged side chains;
  • Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.
  • a person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.
  • the applicant continued to explore the above active sites and found that when the active sites mutated to different amino acids, the corresponding protein activities were also different, and specific mutations could enhance the transaminase activity. Through experimental exploration, it was found that the above specific mutations to the active sites could obtain proteins with enhanced activity.
  • the amino acid mutation sites of the transaminase protein flexible selection and combination can be made in the above mutations.
  • the mutation of the transaminase mutant includes any of the following amino acid mutations:
  • a DNA molecule which encodes the above-mentioned transaminase mutant.
  • a recombinant plasmid is provided, wherein the recombinant plasmid is connected to the above-mentioned DNA molecule.
  • the DNA can encode the aminotransferase mutant and can be connected to a recombinant plasmid to form a circular DNA. Both the DNA and the recombinant plasmid can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the aminotransferase mutant.
  • a host cell wherein the above-mentioned recombinant plasmid is transformed.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell can be Escherichia coli
  • the eukaryotic cell can be yeast.
  • the recombinant plasmid can be replicated in the host cells, and the DNA molecules carried on the recombinant plasmid can also be transcribed and translated to obtain a large number of transaminase mutants.
  • the host cells can be crushed for protein purification, crushed for crude enzyme catalysis or other methods to obtain transaminase mutants, and then catalyze amine compounds.
  • the host cell is a non-plant-derived host cell.
  • a method for preparing a chiral amine compound comprising using the above-mentioned aminotransferase mutant to perform a transamination reaction on a ketone substrate shown in formula I under the action of an amino donor to prepare a chiral amine compound;
  • Ar1 is selected from a first substituted aryl, a first unsubstituted aryl, a substituted arylene or an unsubstituted arylene, a substituted heteroarylene or an unsubstituted heteroarylene;
  • Ar2 is selected from a second substituted aryl, a second unsubstituted aryl, a substituted cycloalkyl or an unsubstituted cycloalkyl, an alkyl or an alkylene;
  • R is selected from H, an alkyl, an alkylene or an alkylene group, the number of C atoms of the alkyl, the alkylene or the alkylene
  • the above transaminase mutant is used to perform a transamination reaction on the ketone compound shown in formula I under the action of an amino donor to obtain a chiral amine compound.
  • the above transaminase mutant can perform chiral catalysis on the above ketone compound to synthesize the required large sterically hindered chiral amine.
  • the activity and tolerance of the transaminase are greatly improved, so that it can be catalyzed under industrial production conditions such as extreme environments, which can improve production efficiency and reduce industrial production costs.
  • the substituents of the first substituted aryl group, the second substituted aryl group or the substituted arylene group are each independently selected from halogen.
  • the substituents are independently located at any one or more positions of the ortho, meta or para position of the first substituted aryl group, the second substituted aryl group or the substituted arylene group.
  • the halogen is selected from F, Cl or Br.
  • Ar1 is selected from unsubstituted heteroarylene
  • Ar2 is selected from a second substituted aryl
  • the substituent of the second substituted aryl is selected from halogen
  • R is selected from a substituted alkylene
  • the number of C atoms of the substituted alkylene is selected from 1 to 5
  • the substituent is selected from hydroxyl
  • the substituted alkylene is connected to Ar1 to form a ring.
  • Ar1 is selected from a first substituted aromatic group or a first unsubstituted aromatic group, the substituent of the first substituted aromatic group is selected from methyl or Cl, Ar2 is selected from an unsubstituted cycloalkyl group, an alkyl group or a second unsubstituted aromatic group, the number of C atoms of the unsubstituted cycloalkyl group is selected from 3 to 5, the alkyl group is selected from isopropyl or ethyl, R is selected from an alkyl group, and the alkyl group is selected from isopropyl, methyl or ethyl.
  • Ar1 is selected from a first substituted aryl group or a first unsubstituted aryl group
  • the substituent of the first substituted aryl group is selected from methyl or -CH2CH2OH
  • Ar2 is selected from a second unsubstituted aryl group or an alkyl group
  • the alkyl group is selected from methyl or isopropyl
  • R is selected from a substituted alkylene group, the number of C atoms of the substituted alkylene group is selected from 1 to 5, the substituent is selected from hydroxyl, and the substituted alkylene group is connected to Ar1 to form a ring.
  • the ketone compound is selected from:
  • the inventors conducted homology modeling on the selected mutants based on the resolved three-dimensional structure of the protein (PDB: 4BA5), conducted molecular docking on different large sterically hindered ketone compounds based on the model structure, and under the guidance of the above-mentioned strategy for improving protein stability, selected 19 residues that may affect the catalytic activity and stability of the protein for modification, including: S27, V31, R34, M64, A74, R77, T87, I91, S101, Y116, T117, V124, N151, L213, T285, A286, V315, C418 and L449.
  • the protein modification methods include saturation mutation and combined mutation.
  • Saturation mutagenesis is a method of modifying the coding gene of the target protein to obtain mutants in which the amino acids at the target site are replaced by 19 other amino acids in a short period of time. This method is not only a powerful tool for protein-directed modification, but also an important means for studying protein structure-function relationships. Saturation mutagenesis often achieves more ideal evolution than single-point mutagenesis. These problems that site-directed mutagenesis cannot solve are precisely the unique features of saturation mutagenesis. Saturation mutagenesis is constructed by whole plasmid PCR, and then the PCR product is digested with DPNI enzyme to remove the template and transformed into Escherichia coli BL21 (DE3). Screening of mutants often requires high-throughput screening methods. Therefore, we developed the following method to screen mutant tolerance.
  • Mutant culture Add 300 ⁇ L LB medium to each well of a 96-well plate, inoculate the single clone on the agar plate into a deep-well 96-well plate, and culture overnight at 37°C and 200 rpm; use Qpix to transfer the overnight cultured bacterial solution to another 96-well plate, add 800 ⁇ L LB medium to each well, and culture at 37°C and 200 rpm for 5 hours.
  • mutants with improved traits were obtained. Then, the optimal mutant was induced in a 2L shake flask (the best conditions for induction expression: 25°C, 0.2mM IPTG overnight induction), and after centrifugation to obtain bacterial sludge, the crude enzyme solution was obtained by ultrasonic cell disruption, and finally a g-scale reaction was performed to confirm the optimal mutant's traits. Saturation mutations at a single site often have little effect, so multiple rounds of iterative saturation mutations are generally required to obtain mutants with greatly improved traits.
  • the beneficial amino acid sites obtained by screening can be combined to obtain mutants with better traits.
  • the construction method of double-point mutations in combined mutations is the same as that of single-point mutations, and is constructed by whole-plasmid PCR. Multi-point mutations that mutate 2 or more sites at the same time are amplified by overlapping extension PCR to obtain mutant genes containing multiple point mutations. After restriction endonuclease digestion at both ends, they are connected to the expression vector, transformed into Escherichia coli cells, and coated in LB culture dishes containing 100 ⁇ g/mL ampicillin. Cultured overnight at 37°C to obtain combined mutants. After the above-mentioned combined mutants are correctly identified by sequencing, the 2L shake flask induction culture and reaction verification as described above are carried out.
  • Examples 1-4 show changes in enzyme stability by conversion rates at different methanol concentrations and temperatures.
  • 1 mL reaction system includes 30 mg substrate 1 or substrate 2, 300 ⁇ L methanol, 1 mg PLP, 2 mg isopropylamine hydrochloride, 300 ⁇ L crude enzyme solution (prepared from 30 mg wet bacterial mud), pH 8.0 100 mM phosphate buffer, and reaction at 40 °C for 18 h.
  • + represents a conversion rate less than 10%
  • ++ represents a conversion rate greater than or equal to 10% and less than or equal to 20%.
  • +++ represents a conversion rate greater than or equal to 20% and less than or equal to 30%
  • ++++ represents a conversion rate greater than or equal to 30% and less than or equal to 40%
  • +++++ represents a conversion rate greater than or equal to 40% and less than or equal to 50%
  • ++++++ represents a conversion rate greater than or equal to 50% and less than or equal to 60%.
  • reaction system included 30 mg substrate 1 or substrate 2, 400 ⁇ L methanol, 1 mg PLP, 2 mg isopropylamine hydrochloride, 300 ⁇ L crude enzyme solution (prepared from 30 mg wet bacterial mud), pH 8.0 100 mM phosphate buffer, and reaction at 40 °C for 18 h.
  • + represents a conversion rate less than 10%
  • ++ represents a conversion rate greater than or equal to 10% and less than or equal to 20%.
  • +++ represents a conversion rate greater than or equal to 20% and less than or equal to 30%
  • ++++ represents a conversion rate greater than or equal to 30% and less than or equal to 40%
  • +++++ represents a conversion rate greater than or equal to 40% and less than or equal to 50%
  • ++++++ represents a conversion rate greater than or equal to 50% and less than or equal to 60%.
  • reaction system included 30 mg substrate 1 or substrate 2, 500 ⁇ L methanol, 1 mg PLP, 2 mg isopropylamine hydrochloride, 150 ⁇ L crude enzyme solution (prepared from 15 mg wet bacterial mud), pH 8.0 100 mM phosphate buffer, and reaction at 40 °C for 18 h.
  • + represents a conversion rate less than 10%
  • ++ represents a conversion rate greater than or equal to 10% and less than or equal to 20%.
  • +++ represents a conversion rate greater than or equal to 20% and less than or equal to 30%
  • ++++ represents a conversion rate greater than or equal to 30% and less than or equal to 40%
  • +++++ represents a conversion rate greater than or equal to 40% and less than or equal to 50%
  • ++++++ represents a conversion rate greater than or equal to 50% and less than or equal to 60%.
  • reaction system included 50 mg substrate 1 or substrate 2, 600 ⁇ L methanol, 1 mg PLP, 2 mg isopropylamine hydrochloride, 100 ⁇ L crude enzyme solution (prepared from 10 mg wet bacterial mud), pH 8.0 100 mM phosphate buffer, and reaction at 50 °C for 18 h.
  • + represents a conversion rate less than 10%
  • ++ represents a conversion rate greater than or equal to 10% and less than or equal to 20%.
  • +++ represents a conversion rate greater than or equal to 20% and less than or equal to 30%
  • ++++ represents a conversion rate greater than or equal to 30% and less than or equal to 40%
  • +++++ represents a conversion rate greater than or equal to 40% and less than or equal to 50%
  • ++++++ represents a conversion rate greater than or equal to 50% and less than or equal to 60%.
  • 1mL reaction system includes 50mg substrate 1 or substrate 2, 1mg PLP, 2mg isopropylamine hydrochloride, 100 ⁇ L crude enzyme solution (prepared from 10mg wet bacterial mud) treated at 70°C for 1h or treated with methanol, ethanol, acetonitrile, ethyl acetate, dichloromethane, tert-methyl ether and n-heptane for 1h, and the control is the same amount of enzyme solution without any treatment, pH8.0 100mM phosphate buffer, and reaction at 40°C for 18h.
  • relative residual activity refers to the percentage of enzyme activity measured after the enzyme solution has been treated with extreme conditions such as high temperature and organic solvents, compared with the enzyme activity of the enzyme solution under optimal conditions without being treated under extreme conditions. Under the same treatment conditions, the higher the relative residual activity, the higher the stability of the enzyme under these conditions.
  • * represents an activity that is 0.1-0.5 times higher than the parent; ** represents an activity that is 0.5-1.0 times higher than the parent; *** represents an activity that is 1.0-1.5 times higher than the parent; **** represents an activity that is 1.5-2 times higher than the parent.
  • + represents the relative residual activity greater than or equal to 10% and less than 30%
  • ++ represents the relative residual activity greater than or equal to 30% and less than 50%
  • +++ represents the relative residual activity greater than or equal to 50% and less than 70%
  • ++++ represents the relative residual activity greater than or equal to 70% and less than 90%.
  • mutants with improved activity and stability were obtained, which can be used to synthesize a variety of large sterically hindered chiral amines under high temperature and high organic solvent conditions without the need for heavy metal catalysts and toxic reagents, thus achieving green chemistry.
  • the aminotransferase mutant has high biocatalytic activity, stable enzyme, high reaction substrate concentration, high product yield, greatly reduces three wastes, and saves production costs.

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Abstract

提供了一种转氨酶突变体及应用。转氨酶突变体，包括(a)具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(b)在(a)中的氨基酸序列的如下至少一个位点：V315、I91、V124、Y116、A286、C418、T87、S301、S27、V31、R34、M64、A74、R77、S101、T117、N151、L213、T285、T107或L449，经过氨基酸突变且具有转氨酶活性功能的蛋白质；(c)与(a)和(b)中任一项限定的氨基酸序列具有80％以上同源性且具有转氨酶功能的蛋白质。解决了现有技术中转氨酶在催化大位阻手性胺化合物的工业化生产中活性或耐受性较差的问题。

Description

转氨酶突变体及应用
本申请是以CN申请号为202211538995.8，申请日为2022年12月2日的中国申请为基础，并主张其优先权，该CN申请的公开内容再次作为整体引入本申请中。
技术领域
本发明涉及酶催化领域，具体而言，涉及一种转氨酶突变体及应用。
背景技术
手性胺是许多重要生物活性分子的结构单元和合成很多手性药物的关键中间体。目前，主要有三种方法可以用于手性胺的合成，包括化学法、生物拆分法和生物不对称合成法。其中，化学法中存在反应路线较长、且条件苛刻，存在合成中使用有毒的过渡态金属催化剂、产品立体选择性低、产率低等缺点；而生物拆分法的理论最高收率只有50％，这两种方法在放大生产上都有存在一定的局限性。
转氨酶催化的不对称合成手性胺，由于具有高选择性、高转化率、反应条件温和以及环境友好等优点，越来越受到人们的关注，现在已成为广泛使用的一种手性胺的制备方法。然而，大多数野生型转氨酶底物范围有限，通常难以合成大位阻手性胺化合物(指催化底物的羰基旁边一侧的取代基大于甲基)。CN114875006A公开了一种转氨酶突变体可以高效合成手性胺尤其是大位阻手性胺。但这些突变体在工业化应用时稳定性相对较差，很难适应放大工艺中的高温、高浓度有机溶剂等极端环境。
通过酶进化的方法可以提高转氨酶的耐受性。Cao，J.，et al对来源于土曲霉的(R)-型转氨酶进行热稳定性改造，通过定点突变构建了13个突变体并通过实验确认了3个突变体(E133Q、D224K和E253A)有助于酶热稳定的提高，其中突变体D224K在40℃下的稳定性提高了4.23倍(Front.Chem.2021，9：664156.)。另外，Cai，B.，et al.利用来源于烟曲霉Af293的(R)-型转氨酶来构建高效生产(R)-α-苯乙胺的酶法工艺，通过定向进化得到的突变体活性增加了3000倍以上，对异丙胺(2M)的耐受性也大大提高，并且在工业试验规模中实现了高达168g L-1d-1的(R)-α-苯乙胺产量(Org.Process Res.Dev.2022，26，7，2004-2012)。
因此，为了进一步适应合成大位阻手性胺的工业化生产要求，我们可以通过酶进化的方法，提高转氨酶在极端条件下的稳定性。以进一步促进转氨酶的固定化和连续化应用，提高生产效率，降低工业生产成本，减少工业三废排放。
发明内容
本发明的主要目的在于提供一种转氨酶突变体及应用，以解决现有技术中转氨酶在催化大位阻手性胺化合物的工业化生产中活性或耐受性较差的问题。
为了实现上述目的，根据本发明的第一个方面，提供了一种转氨酶突变体，包括(a)具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(b)在(a)中的氨基酸序列的如下至少一个位点：V315、I91、V124、Y116、A286、C418、T87、S301、S27、V31、R34、M64、A74、R77、S101、T117、N151、L213、T285、T107或L449，经过氨基酸突变且具有转氨酶活性功能的蛋白质；(c)与(a)和(b)中任一项限定的氨基酸序列具有80％以上同源性且具有转氨酶功能的蛋白质。
进一步地，(b)的氨基酸突变各自独立地选自如下：V315T或V315R或V315D或V315A或V315K或V315H；I91Q或I91M或I91N或I91G或I91F或I91D；V124C或V124S或V124Y或V124P或V124M或V124A或V124I；Y116F或Y116Y或Y116A或Y116R或Y116S或Y116G或Y116H或Y116L；A286I或A286R或A286Q或A286F或A286D或A286M或A286I；C418L或C418W或C418R或C418D或C418F或C418A；T87A或T87Y或T87N或T87V或T87F或T87E；S301A或S301G或S301H或S301K或S301W或S301I或S301M或S301N；S27A或S27V或S27N或S27M或S27E或S27R或S27；S101A或S101R或S101Y或S101H；R34H或R34K或R34N或R34T或R34F或R34M；T117V或T117M或T117L或T117T或T117R或T117S；T285M或T285Y或T285I或T285A或T285G；T107S或T107Q或T107A或T107G；其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸；优选地，(c)中，与(a)或(b)中限定的氨基酸序列具有85％以上，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有转氨酶功能的蛋白质。
进一步地，转氨酶突变体的突变包括如下任意一种氨基酸突变：V315T；V315R；V315D；V315A；V315K；V315H；V315T+I91Q；V315T+I91M；V315T+I91N；V315T+I91G；V315T+I91F；V315T+I91D；V315T+V124C；V315T+V124S；V315T+V124Y；V315T+V124P；V315T+V124M；V315T+V124A；V315T+V124C+I91N；V315T+V124C+I91Q；V315T+V124C+I91M；V315T+V124C+I91G；V315T+V124C+I91F；V315T+I91Q+V124S；V315T+I91Q+V124I；V315T+I91Q+V124P；V315T+I91Q+V124M；V315T+I91Q+V124A；V315T+I91Q+V124Y；V315T+I91Q+V124S+Y116F；V315T+I91Q+V124S+Y116Y；V315T+I91Q+V124S+Y116A；V315T+I91Q+V124S+Y116R；V315T+V124C+I91N+Y116F；V315T+V124C+I91N+Y116A；V315T+V124C+I91N+Y116S；V315T+V124C+I91N+Y116G；V315T+V124C+I91N+Y116H；V315T+V124C+I91N+Y116L；V315T+V124C+I91N+Y116F+A286I；V315T+V124C+I91N+Y116F+A286R；V315T+V124C+I91N+Y116F+A286Q；V315T+V124C+I91N+Y116F+A286F；V315T+V124C+I91N+Y116F+A286D；V315T+V124C+I91N+Y116F+A286M；V315T+V124C+I91N+Y116F+A286I+C418L；V315T+V124C+I91N+Y116F+A286I+C418W；V315T+V124C+I91N+Y116F+A286I+C418R；V315T+V124C+I91N+Y116F+A286I+C418D；V315T+V124C+I91N+Y116F+A286I+C418F；V315T+V124C+I91N+Y116F+A286I+C418A；V315T+V124C+I91N+Y116F+A286I+T87A； V315T+V124C+I91N+Y116F+A286I+T87Y；V315T+V124C+I91N+Y116F+A286I+T87N；V315T+V124C+I91N+Y116F+A286I+T87V；V315T+V124C+I91N+Y116F+A286I+T87F；V315T+V124C+I91N+Y116F+A286I+T87E；V315T+V124C+I91N+Y116F+A286I+T87A+S301A；V315T+V124C+I91N+Y116F+A286I+T87A+S301G；V315T+V124C+I91N+Y116F+A286I+T87A+S301H；V315T+V124C+I91N+Y116F+A286I+T87A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S301W；V315T+V124C+I91N+Y116F+A286I+T87A+S27A；V315T+V124C+I91N+Y116F+A286I+T87A+S27V；V315T+V124C+I91N+Y116F+A286I+T87A+S27N；V315T+V124C+I91N+Y116F+A286I+T87A+S27M；V315T+V124C+I91N+Y116F+A286I+T87A+S27E；V315T+V124C+I91N+Y116F+A286I+T87A+S27R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I； V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117S；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285G；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G。
为了实现上述目的，根据本发明的第二个方面，提供了一种DNA分子，该DNA分子编码上述转氨酶突变体。
为了实现上述目的，根据本发明的第三个方面，提供了一种重组质粒，该重组质粒连接有上述DNA分子。
为了实现上述目的，根据本发明的第四个方面，提供了一种宿主细胞，该宿主细胞内转化有上述重组质粒。
为了实现上述目的，根据本发明的第五个方面，提供了一种手性胺化合物的制备方法，该制备方法包括利上述的转氨酶突变体在氨基供体的作用下，对式I所示酮类底物进行转氨基反应，得到手性胺化合物，
Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基；Ar2选自第二取代芳基、第二未取代芳基、取代环烷基或未取代环烷基、烷基或者亚烷基；R选自H、烷基、亚烷基或次烷基，烷基、亚烷基或次烷基的C原子数选自1～5，烷基、亚烷基或次烷基包括取代烷基、亚烷基或次烷基或未取代烷基、亚烷基或次烷基；当R选自烷基、亚烷基或次烷基时，烷基、亚烷基或次烷基与Ar1和/或Ar2连接成环；第一 取代芳基、取代亚芳基、取代杂亚芳基、第二取代芳基或取代烷基、亚烷基或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH；取代杂亚芳基中的杂原子选自N、O或S。
进一步地，第一取代芳基、第二取代芳基或取代亚芳基的取代基各自独立选自卤素。
进一步地，取代基各自独立位第一取代芳基、第二取代芳基或取代亚芳基的邻位、间位或对位中的任意一个或多个位置。
进一步地，卤素选自F、Cl或Br。
进一步地，Ar1选自未取代杂亚芳基，Ar2选自第二取代芳基，第二取代芳基的取代基选自卤素，R选自取代亚烷基，取代亚烷基的C原子数选自1～5，取代基选自羟基，取代亚烷基与Ar1连接成环。
进一步地，Ar1选自第一取代芳基、或第一未取代芳基，第一取代芳基的取代基选自甲基或者Cl，Ar2选自未取代环烷基、烷基或第二未取代芳基，未取代环烷基的C原子数选自3～5，烷基选自异丙基或乙基，R选自烷基，烷基选自异丙基、甲基或乙基。
进一步地，Ar1选自第一取代芳基、或第一未取代芳基，第一取代芳基的取代基选自甲基或者-CH2CH2OH，Ar2选自第二未取代芳基或烷基，烷基选自甲基或者异丙基，R选自取代亚烷基，取代亚烷基的C原子数选自1～5，取代基选自羟基，取代亚烷基与Ar1连接成环。
进一步地，酮类底物选自
应用本发明的技术方案，以源于Chromobacterium violaceum的转氨酶突变体(SEQ ID NO：1)为母本，进行了饱和突变、组合突变等蛋白质工程改造，获得了在催化大位阻手性胺的反应中，活性和耐受性都得到极大提升的转氨酶突变体。
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。
术语解释：
大位阻手性化合物：本申请中指前手性羰基旁边的基团大于甲基的酮类化合物，其中，大于甲基的基团可以是乙基、丙级、叔丁基或者是苯基叔丁基等。
如背景技术所提到的，手性胺的化学合成与生物拆分方法存在多种不足，利用生物不对称法合成手性胺可具有更高效率的同时，其反应条件也更加温和。但由于大多数野生型转氨酶底物范围有限，因而在合成大位阻手性胺化合物时，转氨酶多存在活性低、耐受性差的问题，而无法进行放大工艺生产。
因此，在本申请中发明人尝试通过酶进化的方法对转氨酶进行改造，进而提高转氨酶在极端条件下的各种性质，使得其在工业化生产中可以提高生产效率，因而提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中，提供了一种转氨酶突变体，包括：(a)具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或(b)在(a)中的氨基酸序列的如下至少一个位点：V315、I91、V124、Y116、A286、C418、T87、S301、S27、V31、R34、M64、A74、R77、S101、T117、N151、L213、T285、T107或L449，经过氨基酸突变且具有转氨酶活性功能的蛋白质；(c)与(a)和(b)中任一项限定的氨基酸序列具有80％以上同源性且具有转氨酶功能的蛋白质。
上述SEQ ID NO：1所示的氨基酸序列，是来源于Chromobacterium violaceum的转氨酶突变体。通过对该氨基酸序列进行同源建模的计算机模拟分析模型结构与不同的大位阻酮化合 物进行分子对接，发现了19个氨基酸残基包括：S27、V31、R34、M64、A74、R77、T87、I91、S101、Y116、T117、V124、N151、L213、T285、A286、V315、C418和L449。这些氨基酸位点有可能影响蛋白催化活性和稳定性。通过对上述氨基酸位点进行突变，能够获得具有转氨酶功能、乃至转氨酶功能增强的蛋白质。对于上述获得的蛋白质，在非关键突变位点和活性位点处可以进行变化，能够获得与上述氨基酸序列有80％以上同源性、且具有转氨酶功能的蛋白质。
SEQ ID NO：1的序列如下：
在一种优选的实施例中，(b)的氨基酸突变各自独立地选自如下：V315T或V315R或V315D或V315A或V315K或V315H；I91Q或I91M或I91N或I91G或I91F或I91D；V124C或V124S或V124Y或V124P或V124M或V124A或V124I；Y116F或Y116Y或Y116A或Y116R或Y116S或Y116G或Y116H或Y116L；A286I或A286R或A286Q或A286F或A286D或A286M或A286I；C418L或C418W或C418R或C418D或C418F或C418A；T87A或T87Y或T87N或T87V或T87F或T87E；S301A或S301G或S301H或S301K或S301W或S301I或S301M或S301N；S27A或S27V或S27N或S27M或S27E或S27R或S27；S101A或S101R或S101Y或S101H；R34H或R34K或R34N或R34T或R34F或R34M；T117V或T117M或T117L或T117T或T117R或T117S；T285M或T285Y或T285I或T285A或T285G；T107S或T107Q或T107A或T107G；其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸；优选地，(c)中，与(a)或(b)中限定的氨基酸序列具有85％以上，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有转氨酶功能的蛋白质。
如本文所用，氨基酸残基缩写如下：丙氨酸(Ala；A)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、精氨酸(Arg；R)、半胱氨酸(Cys；C)、谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)，丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
取代、替换等规则，一般情况下，哪些氨基酸性质类似，替换后的效果也类似。例如，在上述同源蛋白中，可发生保守的氨基酸替换。“保守的氨基酸替换”包括但不限于：
疏水性氨基酸(Ala、Cys、Gly、Pro、Met、Val、Ile、Leu)被其他疏水性氨基酸取代；
侧链粗大的疏水性氨基酸(Phe、Tyr、Trp)被其他侧链粗大的疏水性氨基酸取代；
侧链带正电的氨基酸(Arg、His、Lys)被其他侧链带正电的氨基酸取代；
侧链有极性不带电的氨基酸(Ser、Thr、Asn、Gln)被其他侧链有极性不带电的氨基酸取代。
本领域技术人员也可以根据现有技术中的“blosum62评分矩阵”等本领域技术人员熟知的氨基酸替换规则对氨基酸进行保守替换。
在本申请中，申请人对上述活性位点继续进行探究，发现活性位点突变为不同氨基酸，相应的蛋白质活性也有差别，特定的突变能够增强转氨酶活性。通过试验探究，发现对于活性位点进行上述特定的突变，能够获得活性增强的蛋白质。对于转氨酶蛋白质的氨基酸突变位点，可以在上述的突变中进行灵活的选择和组合。
在一种优选的实施例中，转氨酶突变体的突变包括如下任意一种氨基酸突变：
V315T；V315R；V315D；V315A；V315K；V315H；V315T+I91Q；V315T+I91M；V315T+I91N；V315T+I91G；V315T+I91F；V315T+I91D；V315T+V124C；V315T+V124S；V315T+V124Y；V315T+V124P；V315T+V124M；V315T+V124A；V315T+V124C+I91N；V315T+V124C+I91Q；V315T+V124C+I91M；V315T+V124C+I91G；V315T+V124C+I91F；V315T+I91Q+V124S；V315T+I91Q+V124I；V315T+I91Q+V124P；V315T+I91Q+V124M；V315T+I91Q+V124A；V315T+I91Q+V124Y；V315T+I91Q+V124S+Y116F；V315T+I91Q+V124S+Y116Y；V315T+I91Q+V124S+Y116A；V315T+I91Q+V124S+Y116R；V315T+V124C+I91N+Y116F；V315T+V124C+I91N+Y116A；V315T+V124C+I91N+Y116S；V315T+V124C+I91N+Y116G；V315T+V124C+I91N+Y116H；V315T+V124C+I91N+Y116L；V315T+V124C+I91N+Y116F+A286I；V315T+V124C+I91N+Y116F+A286R；V315T+V124C+I91N+Y116F+A286Q；V315T+V124C+I91N+Y116F+A286F；V315T+V124C+I91N+Y116F+A286D；V315T+V124C+I91N+Y116F+A286M；V315T+V124C+I91N+Y116F+A286I+C418L；V315T+V124C+I91N+Y116F+A286I+C418W；V315T+V124C+I91N+Y116F+A286I+C418R；V315T+V124C+I91N+Y116F+A286I+C418D；V315T+V124C+I91N+Y116F+A286I+C418F；V315T+V124C+I91N+Y116F+A286I+C418A；V315T+V124C+I91N+Y116F+A286I+T87A；V315T+V124C+I91N+Y116F+A286I+T87Y；V315T+V124C+I91N+Y116F+A286I+T87N；V315T+V124C+I91N+Y116F+A286I+T87V；V315T+V124C+I91N+Y116F+A286I+T87F；V315T+V124C+I91N+Y116F+A286I+T87E；V315T+V124C+I91N+Y116F+A286I+T87A+S301A；V315T+V124C+I91N+Y116F+A286I+T87A+S301G；V315T+V124C+I91N+Y116F+A286I+T87A+S301H；V315T+V124C+I91N+Y116F+A286I+T87A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S301W； V315T+V124C+I91N+Y116F+A286I+T87A+S27A；V315T+V124C+I91N+Y116F+A286I+T87A+S27V；V315T+V124C+I91N+Y116F+A286I+T87A+S27N；V315T+V124C+I91N+Y116F+A286I+T87A+S27M；V315T+V124C+I91N+Y116F+A286I+T87A+S27E；V315T+V124C+I91N+Y116F+A286I+T87A+S27R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117S；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M； V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285G；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107A；V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G。
上述氨基酸突变均在本申请实施例中进行试验探究，均具有转氨酶活性，相较于具有SEQ ID NO：1所示的氨基酸序列的母本，能够获得具有稳定性较高、高耐受极端环境、酶活性高，可用于工业放大生产的转氨酶突变体。
在本申请第二种典型的实施方式中，提供了一种DNA分子，该DNA分子编码上述转氨酶突变体。
在本申请第三种典型的实施方式中，提供了一种重组质粒，该重组质粒连接有上述DNA分子。
上述DNA能够编码上述转氨酶突变体，并能够连接在重组质粒上形成环状DNA。上述DNA和重组质粒均能在RNA聚合酶、核糖体、tRNA等的作用下，进行转录、翻译，获得上述转氨酶突变体。
在本申请第四种典型的实施方式中，提供了一种宿主细胞，该宿主细胞内转化有上述重组质粒。宿主细胞可以是原核细胞，也可以是真核细胞。具体地，原核细胞可以是大肠杆菌，真核细胞可以是酵母菌。
利用上述宿主细胞，能够在宿主细胞中进行重组质粒的复制，也能够将重组质粒上携带的DNA分子进行转录、翻译，获得大量转氨酶突变体。利用现有技术，对宿主细胞进行破碎蛋白纯化、破碎后粗酶催化或其他方式，能够获得转氨酶突变体，并进行后续对于胺类化合物的催化。该宿主细胞为非植物来源的宿主细胞。
在本申请第五种典型的实施方式中，提供了一种手性胺化合物的制备方法，该制备方法包括利用上述转氨酶突变体在氨基供体的作用下，对式I所示的酮类底物进行转氨基反应，制备获得手性胺化合物；Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基；Ar2选自第二取代芳基、第二未取代芳基、取代环烷基或未取代环烷基、烷基或者亚烷基；R选自H、烷基、亚烷基或次烷基，烷基、亚烷基或次烷基的C原子数选自1～5，烷基、亚烷基或次烷基包括取代烷基、亚烷基或次烷基或未取代烷基、亚烷基或次烷基；当R选自烷基、亚烷基或次烷基时，烷基、亚烷基或次烷基与Ar1和/或Ar2连接成环；第一取代芳基、取代亚芳基、取代杂亚芳基、第二取代芳基或取代烷基、亚烷基 或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH；取代杂亚芳基中的杂原子选自N、O或S。
利用上述制备方法，利用上述转氨酶突变体在氨基供体的作用下对式I所示的酮类化合物进行转氨基反应，获得手性胺化合物。上述转氨酶突变体，能够对于上述的酮类化合物，进行手性催化，合成所需的大位阻手性胺。大幅提高了转氨酶的活性与耐受性，使其能够在极端环境等工业化生产条件下进行催化，能够提高生产效率，降低工业生产成本。
在一种优选的实施例中，第一取代芳基、第二取代芳基或取代亚芳基的取代基各自独立选自卤素。
在一种优选的实施例中，取代基各自独立位第一取代芳基、第二取代芳基或取代亚芳基的邻位、间位或对位中的任意一个或多个位置。
在一种优选的实施例中，卤素选自F、Cl或Br。
在一种优选的实施例中，Ar1选自未取代杂亚芳基，Ar2选自第二取代芳基，第二取代芳基的取代基选自卤素，R选自取代亚烷基，取代亚烷基的C原子数选自1～5，取代基选自羟基，取代亚烷基与Ar1连接成环。
在一种优选的实施例中，Ar1选自第一取代芳基、或第一未取代芳基，第一取代芳基的取代基选自甲基或者Cl，Ar2选自未取代环烷基、烷基或第二未取代芳基，未取代环烷基的C原子数选自3～5，烷基选自异丙基或乙基，R选自烷基，烷基选自异丙基、甲基或乙基。
在一种优选的实施例中，Ar1选自第一取代芳基、或第一未取代芳基，第一取代芳基的取代基选自甲基或者-CH2CH2OH，Ar2选自第二未取代芳基或烷基，烷基选自甲基或者异丙基，R选自取代亚烷基，取代亚烷基的C原子数选自1～5，取代基选自羟基，取代亚烷基与Ar1连接成环。
在一种优选地实施例中，酮类化合物选自：
本申请中，发明人根据已经解析出的蛋白质的三维结构(PDB：4BA5)，对筛选出的突变体进行同源建模，根据模型结构对不同的大位阻酮化合物进行分子对接，并在上述提高蛋白稳定性策略的指导下，选择了19个有可能影响蛋白催化活性和稳定性的残基进行改造，这些残基包括：S27、V31、R34、M64、A74、R77、T87、I91、S101、Y116、T117、V124、N151、L213、T285、A286、V315、C418和L449。蛋白质改造的方式包括饱和突变和组合突变。
饱和突变是通过对目的蛋白的编码基因进行改造，短时间内获取靶位点氨基酸分别被其它19种氨基酸替代的突变体的一种方法。此方法不仅是蛋白质定向改造的强有力工具，而且是蛋白质结构-功能关系研究的重要手段。饱和突变往往能获得比单点突变更为理想的进化 体。而对于定点突变方法不能解决的这些问题，恰恰是饱和突变方法所擅长的独特之处。饱和突变的构建采用全质粒PCR的方法，之后将PCR产物进行DPNI酶的消化以去除模板，转化大肠杆菌BL21(DE3)。突变体的筛选往往需要高通量的筛选方法，因此，我们开发如下方法进行突变体耐受性的筛选。
开发如下的高通量筛选方法对突变体库进行筛选：
1、突变体培养：96孔板每孔加入300μL LB培养基，将琼脂平板上的单克隆接种到深孔96孔板中，37℃、200rpm过夜培养；利用Qpix转接过夜培养的菌液，至另一块每孔加入800μL LB培养基96孔板中，37℃、200rpm，培养5h后，待96孔板菌液OD600达到0.6-0.9，再次利用Qpix向96孔板中加入IPTG溶液，使孔板中IPTG终浓度为0.1mM，25℃、200rpm过夜诱导约16h；4000rpm，离心5min，弃上清，用全细胞进行反应。
2、96孔板高通量筛选体系：
将8μL PLP母液(1mg/mL)、3.5μL的6M异丙胺盐酸盐(20eq)，0.1M Tris-Cl 9.0补到100μL混匀，将混样分装至每孔装有菌泥的96孔板中，最后加入底物母液(0.3mg底物溶于不同浓度的甲醇中)，混匀，50℃恒温摇床700rpm，反应18h。之后每孔加入3倍体积的甲醇，离心，上清样送UPLC进行分析。
通过上述的突变体初筛，得到性状提高的突变体。然后，会对最优突变体进行2L摇瓶诱导培养(诱导表达的最佳条件：25℃，0.2mM IPTG过夜诱导)，离心得到菌泥后，通过超声破碎细胞的方法获得粗酶液，最后进行g级规模的反应对最优突变体性状进行确认。单个位点的饱和突变往往提高效果不明显，因此一般需要进行多轮的迭代饱和突变才能获得性状大幅提高的突变体。
在饱和突变获得活性提高的突变体基础上，可对筛选得到的有益的氨基酸位点进行组合，以获得性状更优的突变体。组合突变中双点突变的构建方法和单点突变的构建方法一样，采用全质粒PCR法构建。同时突变2个及以上位点的多点突变通过采用重叠延伸PCR扩增进行，获得含多点突变的突变基因，两端经限制性内切酶酶切后，连接到表达载体上，转化至大肠杆菌细胞内，涂布于含有100μg/mL氨苄青霉素的LB培养皿中，37℃培养过夜，获得组合突变体。上述组合突变体经测序鉴定正确后，进行如上述的2L摇瓶诱导培养和反应验证。
经过多轮进化，得到了一系列转氨酶突变体，结果发现这些突变体在催化大位阻手性胺的反应中，活性和耐受性都得到极大提升。这些突变体被证明可以很好的用在大位阻手性胺的工业化生产中。
以下结合具体实施例对本申请作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。
下列实施例1-4中通过不同甲醇浓度和温度下的转化率表示酶稳定性的变化。
实施例1
在母本SEQ ID NO：1的基础上进行2轮饱和突变，具体突变位点见下表，并按照如下反应条件对突变体的催化活性进行检测。
1mL反应体系包括30mg底物1或底物2，300μL甲醇，1mg PLP，2mg异丙胺盐酸盐，300μL粗酶液(由30mg湿菌泥制得)，pH8.0100mM磷酸盐buffer，40℃反应18h。
检测结果见下表
表1：
注：上表中，+代表转化率小于10％，++代表转化率大于等于10％且小于等于20％。+++代表转化率大于等于20％且小于等于30％，++++代表转化率大于等于30％且小于等于40％，+++++代表转化率大于等于40％且小于等于50％，++++++代表转化率大于等于50％且小于等于60％。
实施例2
在实施例1的基础上继续进行饱和突变和组合突变，并按照如下反应条件对突变进行活性筛选，
1mL反应体系包括30mg底物1或底物2，400μL甲醇，1mg PLP，2mg异丙胺盐酸盐，300μL粗酶液(由30mg湿菌泥制得)，pH8.0 100mM磷酸盐buffer，40℃反应18h。
结果见下表
表2：
注：上表中，+代表转化率小于10％，++代表转化率大于等于10％且小于等于20％。+++代表转化率大于等于20％且小于等于30％，++++代表转化率大于等于30％且小于等于40％，+++++代表转化率大于等于40％且小于等于50％，++++++代表转化率大于等于50％且小于等于60％。
实施例3
在实施例2的基础上进行多轮饱和突变，并按照如下反应条件对突变体的催化活性进行检测：
1mL反应体系包括30mg底物1或底物2，500μL甲醇，1mg PLP，2mg异丙胺盐酸盐，150μL粗酶液(由15mg湿菌泥制得)，pH8.0 100mM磷酸盐buffer，40℃反应18h。
结果见下表
表3：
注：上表中，+代表转化率小于10％，++代表转化率大于等于10％且小于等于20％。+++代表转化率大于等于20％且小于等于30％，++++代表转化率大于等于30％且小于等于40％，+++++代表转化率大于等于40％且小于等于50％，++++++代表转化率大于等于50％且小于等于60％。
实施例4
在实施例3的基础上继续进行多轮饱和突变，并按照如下反应条件对突变体的催化活性进行检测：
1mL反应体系包括50mg底物1或底物2，600μL甲醇，1mg PLP，2mg异丙胺盐酸盐，100μL粗酶液(由10mg湿菌泥制得)，pH8.0 100mM磷酸盐buffer，50℃反应18h。
表4：

注：上表中，+代表转化率小于10％，++代表转化率大于等于10％且小于等于20％。+++代表转化率大于等于20％且小于等于30％，++++代表转化率大于等于30％且小于等于40％，+++++代表转化率大于等于40％且小于等于50％，++++++代表转化率大于等于50％且小于等于60％。
实施例5
选择上述实施例中获得的部分突变体，分别测试高温和不同有机溶剂处理后酶的稳定性，反应按照如下条件进行：
1mL反应体系包括50mg底物1或底物2，1mg PLP，2mg异丙胺盐酸盐，经过在70℃下处理1h或者分别经过甲醇、乙醇、乙腈、乙酸乙酯、二氯甲烷、甲叔醚和正庚烷处理1h后的100μL粗酶液(由10mg湿菌泥制得)，对照为相同量的酶液不经任何处理，pH8.0 100mM磷酸盐buffer，40℃反应18h。
结果见下表
表5-1：
表5-2：
表6-1：

表6-2：
注：1)上表中，相对残余活力是指经高温、有机溶剂等极端条件适量处理后的酶液测定的酶活力，与未经过极端环境下处理的酶液在最适条件下的酶活力的百分比。在相同的处理条件下，相对残余活力高，说明该酶在此条件下的稳定越高。
2)对照中，*代表活性较母本提高了0.1-0.5倍；**代表活性较母本提高了0.5-1.0倍；***代表活性较母本提高了1.0-1.5倍；****代表活性较母本提高了1.5-2倍。
3)+代表相对残余活力大于等于10％且小于30％，++代表相对残余活力大于等于30％且小于50％，+++代表相对残余活力大于等于50％且小于70％，++++代表相对残余活力大于等于70％且小于90％。
由上述实施例描述中，可以看出：
1)本申请中得到的突变体在高温下的稳定性有了明显的提高；
2)对有机溶剂耐受性方面，除了对甲醇的耐受性提高明显外，对于上述6种常用的有机溶剂的耐受性均有不同程度的提高。
3)另外，从上述对照实验看出，酶进化除了提高突变体的稳定性外，酶活方面也有了不同程度的提高。
实施例6
室温向250mL四口瓶内加入20mL 100mmol/L磷酸盐缓冲液，20mL 6mol/L异丙胺盐酸盐溶液(2vol)，60mL甲醇，调pH＝8.5～9.0。再加入0.1g磷酸吡哆醛，5g(底物1)，搅拌均匀，再加入SEQ ID NO：1基础上突变的的转氨酶突变体(V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M)的酶液2mL(0.5g湿菌泥/mL酶液)调pH＝8.5～9.0。升温至50℃搅拌反应，待反应完全后，将体系调酸至pH＝2-3，变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH＝12再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T＜40℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测，纯度＞99％，de值＞99％，收率85％
实施例7
室温向250mL四口瓶内加入20mL 100mmol/L磷酸盐缓冲液，20mL 6mol/L异丙胺盐酸盐溶液(2vol)，60mL甲醇，调pH＝8.5～9.0。再加入0.1g磷酸吡哆醛，5g(底物2)，搅拌均匀，再加入SEQ ID NO：1基础上突变的的转氨酶突变体(V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M)的酶液2mL(0.5g湿菌泥/mL酶液)调pH＝8.5～9.0。升温至50℃搅拌反应，待反应完全后，将体系调酸至pH＝2-3，变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH＝12再用50mL 甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T＜40℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测，纯度＞99％，de值＞99％，收率84％
实施例8
室温向250mL四口瓶内加入20mL 100mmol/L磷酸盐缓冲液，20mL 6mol/L异丙胺盐酸盐溶液(2vol)，60mL甲醇，调pH＝8.5～9.0。再加入0.1g磷酸吡哆醛，5g(底物16)，搅拌均匀，再加入SEQ ID NO：1基础上突变的的转氨酶突变体(V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M)的酶液2mL(0.5g湿菌泥/mL酶液)调pH＝8.5～9.0。升温至50℃搅拌反应，待反应完全后，将体系调酸至pH＝2-3，变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH＝12再用50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T＜40℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测，纯度＞99％，de值＞99％，收率75％
实施例9
室温向250mL四口瓶内加入20mL 100mmol/L磷酸盐缓冲液，20mL 6mol/L异丙胺盐酸盐溶液(2vol)，60mL甲醇，调pH＝8.5～9.0。再加入0.1g磷酸吡哆醛，5g(底物17)，搅拌均匀，再加入SEQ ID NO：1基础上突变的的转氨酶突变体(V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M)的酶液2mL(0.5g湿菌泥/mL酶液)调pH＝8.5～9.0。升温至50℃搅拌反应，待反应完全后，将体系调酸至pH＝2-3，变性蛋白。过滤后滤液用50mL甲基叔丁基醚萃取。水相调pH＝12再用 50mL甲基叔丁基醚萃取2次。合并有机相用无水硫酸镁干燥后于T＜40℃，P≤-0.06Mpa条件下浓缩至无馏分。得到目标产物
经HPLC检测，纯度＞99％，de值＞99％，收率80％
实施例10
采用SEQ ID NO：1基础上突变的转氨酶突变体(V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M)的酶液，参照实施例6-9的催化合成步骤，对底物3-15进行催化反应，结果如下表：
表7：
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：
1)通过对转氨酶进行进化，得到了活性和稳定性都有所提高的突变体，可以在高温、高有机溶剂条件下进行多种大位阻手性胺的合成，不需要重金属催化剂和有毒试剂参与，实现绿色化学。
2)该转氨酶突变体生物催化活性高，酶稳定，反应底物浓度高，产品收率高，大大降低三废，节约生产成本。
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 (14)

  1. 一种转氨酶突变体，其特征在于，包括：
    (a)具有SEQ ID NO：1所示的氨基酸序列的蛋白质；或
    (b)在(a)中的所述氨基酸序列的如下至少一个位点：V315、I91、V124、Y116、A286、C418、T87、S301、S27、V31、R34、M64、A74、R77、S101、T117、N151、L213、T285、T107或L449，经过氨基酸突变且具有转氨酶活性功能的蛋白质；
    (c)与(a)和(b)中任一项限定的所述氨基酸序列具有80％以上同源性且具有转氨酶功能的蛋白质。
  2. 根据权利要求1所述的转氨酶突变体，其特征在于，所述(b)的所述氨基酸突变各自独立地选自如下：
    V315T或V315R或V315D或V315A或V315K或V315H；
    I91Q或I91M或I91N或I91G或I91F或I91D；
    V124C或V124S或V124Y或V124P或V124M或V124A或V124I；
    Y116F或Y116Y或Y116A或Y116R或Y116S或Y116G或Y116H或Y116L；
    A286I或A286R或A286Q或A286F或A286D或A286M或A286I；
    C418L或C418W或C418R或C418D或C418F或C418A；
    T87A或T87Y或T87N或T87V或T87F或T87E；
    S301A或S301G或S301H或S301K或S301W或S301I或S301M或S301N；
    S27A或S27V或S27N或S27M或S27E或S27R或S27；
    S101A或S101R或S101Y或S101H；
    R34H或R34K或R34N或R34T或R34F或R34M；
    T117V或T117M或T117L或T117T或T117R或T117S；
    T285M或T285Y或T285I或T285A或T285G；
    T107S或T107Q或T107A或T107G；
    其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸；
    优选地，所述(c)中，与(a)或(b)中限定的所述氨基酸序列具有85％以上，优选90％以上，更优选95％以上，进一步优选99％以上同源性且具有转氨酶功能的蛋白质。
  3. 根据权利要求2所述的转氨酶突变体，其特征在于，所述转氨酶突变体的突变包括如下任意一种氨基酸突变：
    V315T；
    V315R；
    V315D；
    V315A；
    V315K；
    V315H；
    V315T+I91Q；
    V315T+I91M；
    V315T+I91N；
    V315T+I91G；
    V315T+I91F；
    V315T+I91D；
    V315T+V124C；
    V315T+V124S；
    V315T+V124Y；
    V315T+V124P；
    V315T+V124M；
    V315T+V124A；
    V315T+V124C+I91N；
    V315T+V124C+I91Q；
    V315T+V124C+I91M；
    V315T+V124C+I91G；
    V315T+V124C+I91F；
    V315T+I91Q+V124S；
    V315T+I91Q+V124I；
    V315T+I91Q+V124P；
    V315T+I91Q+V124M；
    V315T+I91Q+V124A；
    V315T+I91Q+V124Y；
    V315T+I91Q+V124S+Y116F；
    V315T+I91Q+V124S+Y116Y；
    V315T+I91Q+V124S+Y116A；
    V315T+I91Q+V124S+Y116R；
    V315T+V124C+I91N+Y116F；
    V315T+V124C+I91N+Y116A；
    V315T+V124C+I91N+Y116S；
    V315T+V124C+I91N+Y116G；
    V315T+V124C+I91N+Y116H；
    V315T+V124C+I91N+Y116L；
    V315T+V124C+I91N+Y116F+A286I；
    V315T+V124C+I91N+Y116F+A286R；
    V315T+V124C+I91N+Y116F+A286Q；
    V315T+V124C+I91N+Y116F+A286F；
    V315T+V124C+I91N+Y116F+A286D；
    V315T+V124C+I91N+Y116F+A286M；
    V315T+V124C+I91N+Y116F+A286I+C418L；
    V315T+V124C+I91N+Y116F+A286I+C418W；
    V315T+V124C+I91N+Y116F+A286I+C418R；
    V315T+V124C+I91N+Y116F+A286I+C418D；
    V315T+V124C+I91N+Y116F+A286I+C418F；
    V315T+V124C+I91N+Y116F+A286I+C418A；
    V315T+V124C+I91N+Y116F+A286I+T87A；
    V315T+V124C+I91N+Y116F+A286I+T87Y；
    V315T+V124C+I91N+Y116F+A286I+T87N；
    V315T+V124C+I91N+Y116F+A286I+T87V；
    V315T+V124C+I91N+Y116F+A286I+T87F；
    V315T+V124C+I91N+Y116F+A286I+T87E；
    V315T+V124C+I91N+Y116F+A286I+T87A+S301A；
    V315T+V124C+I91N+Y116F+A286I+T87A+S301G；
    V315T+V124C+I91N+Y116F+A286I+T87A+S301H；
    V315T+V124C+I91N+Y116F+A286I+T87A+S301K；
    V315T+V124C+I91N+Y116F+A286I+T87A+S301W；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27V；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27N；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27M；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27E；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27R；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101R；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101Y；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101H；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34H；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34K；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34N；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34T；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+R34F；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117V；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+T117M；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301K；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301I；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301G；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S301W；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34T；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34N；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34F；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34M；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301M；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301K；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301I；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301N；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+S301A；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117L；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117T；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117R；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117S；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285M；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285Y；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285I；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T285A；
    V315T+V124C+I9IN+Y116F+A286I+T87A+S2/A+S101A+R34H+T11/V+T285G；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107S；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107Q；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107A；
    V315T+V124C+I91N+Y116F+A286I+T87A+S27A+S101A+R34H+T117V+T107G。
  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，所述重组质粒连接有权利要求4所述的DNA分子。
  6. 一种宿主细胞，其特征在于，所述宿主细胞内转化有权利要求5所述的重组质粒。
  7. 一种手性胺化合物的制备方法，其特征在于，所述制备方法包括利用权利要求1至3中任一项所述的转氨酶突变体在氨基供体的作用下，对式I所示酮类底物进行转氨基反应，得到手性胺化合物，
    Ar1选自第一取代芳基、第一未取代芳基、取代亚芳基或未取代亚芳基、取代杂亚芳基或未取代杂亚芳基；
    Ar2选自第二取代芳基、第二未取代芳基、取代环烷基或未取代环烷基、烷基或者亚烷基；
    R选自H、烷基、亚烷基或次烷基，所述烷基、亚烷基或次烷基的C原子数选自1～5，所述烷基、亚烷基或次烷基包括取代烷基、亚烷基或次烷基或未取代烷基、亚烷基或次烷基；
    当R选自烷基、亚烷基或次烷基时，所述烷基、亚烷基或次烷基与所述Ar1和/或所述Ar2连接成环；
    所述第一取代芳基、所述取代亚芳基、所述取代杂亚芳基、所述第二取代芳基或所述取代烷基、亚烷基或次烷基中的取代基各自独立选自卤素、羟基、氨基、甲基、乙基或-CH2CH2OH；
    所述取代杂亚芳基中的杂原子选自N、O或S。
  8. 根据权利要求7所述的制备方法，其特征在于，所述第一取代芳基、所述第二取代芳基或所述取代亚芳基的所述取代基各自独立选自所述卤素。
  9. 根据权利要求8所述的制备方法，其特征在于，所述取代基各自独立位于所述第一取代芳基、所述第二取代芳基或所述取代亚芳基的邻位、间位或对位中的任意一个或多个位置。
  10. 根据权利要求9所述的制备方法，其特征在于，所述卤素选自F、Cl或Br。
  11. 根据权利要求10所述的制备方法，其特征在于，
    所述Ar1选自所述未取代杂亚芳基，
    所述Ar2选自所述第二取代芳基，所述第二取代芳基的所述取代基选自所述卤素，
    所述R选自所述取代亚烷基，所述取代亚烷基的C原子数选自1～5，所述取代基选自羟基，
    所述取代亚烷基与所述Ar1连接成环。
  12. 根据权利要求10所述的制备方法，其特征在于，
    所述Ar1选自所述第一取代芳基、或所述第一未取代芳基，所述第一取代芳基的所述取代基选自甲基或者Cl，
    所述Ar2选自所述未取代环烷基、烷基或第二未取代芳基，所述未取代环烷基的C原子数选自3～5，所述烷基选自异丙基或乙基，
    所述R选自所述烷基，所述烷基选自异丙基、甲基或乙基。
  13. 根据权利要求10所述的制备方法，其特征在于，
    所述Ar1选自所述第一取代芳基、或所述第一未取代芳基，所述第一取代芳基的所述取代基选自甲基或者-CH2CH2OH，
    所述Ar2选自所述第二未取代芳基或烷基，所述烷基选自甲基或者异丙基，
    所述R选自取代亚烷基，所述取代亚烷基的C原子数选自1～5，所述取代基选自羟基，
    所述取代亚烷基与所述Ar1连接成环。
  14. 根据权利要求10至13中任一项所述的制备方法，其特征在于，所述酮类底物选自
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116083385A (zh) * 2022-12-02 2023-05-09 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116083406A (zh) * 2022-12-02 2023-05-09 凯莱英生命科学技术(天津)有限公司 酰胺酶突变体及其应用
CN118028262B (zh) * 2024-04-11 2024-08-30 天津凯莱英生物科技有限公司 转氨酶突变体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112980810A (zh) * 2021-02-04 2021-06-18 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用
CN114875006A (zh) * 2022-06-21 2022-08-09 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及手性胺化合物的制备方法
WO2022166104A1 (zh) * 2021-02-04 2022-08-11 凯莱英医药集团（天津）股份有限公司 手性胺化合物的合成方法
EP4053269A1 (en) * 2019-10-28 2022-09-07 Asymchem Laboratories (Tianjin) Co., Ltd Transaminase mutant and application thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110832079A (zh) * 2017-05-23 2020-02-21 新加坡国立大学 苯乙醇、醛、酸、胺及相关化合物的生物生产
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
CN110592042B (zh) * 2019-10-28 2021-04-23 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用
CN116083385A (zh) * 2022-12-02 2023-05-09 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及应用
CN117431228A (zh) * 2023-10-27 2024-01-23 浙江大学 一种高立体选择性转氨酶突变体、编码基因及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4053269A1 (en) * 2019-10-28 2022-09-07 Asymchem Laboratories (Tianjin) Co., Ltd Transaminase mutant and application thereof
CN112980810A (zh) * 2021-02-04 2021-06-18 凯莱英医药集团（天津）股份有限公司 转氨酶突变体及其应用
WO2022166104A1 (zh) * 2021-02-04 2022-08-11 凯莱英医药集团（天津）股份有限公司 手性胺化合物的合成方法
CN114875006A (zh) * 2022-06-21 2022-08-09 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及手性胺化合物的制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 1 December 2020 (2020-12-01), "Chain A, Aminotransferase", XP093177104, Database accession no. PDB: 4BA5_A *
DATABASE Protein 1 December 2020 (2020-12-01), "Chain B, Aminotransferase", XP093177108, Database accession no. PDB: 4BA5_B *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116083385A (zh) * 2022-12-02 2023-05-09 凯莱英生命科学技术(天津)有限公司 转氨酶突变体及应用

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