CN116515788A - Novel R-type omega-aminotransferase and application thereof - Google Patents
Novel R-type omega-aminotransferase and application thereof Download PDFInfo
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- CN116515788A CN116515788A CN202310761031.8A CN202310761031A CN116515788A CN 116515788 A CN116515788 A CN 116515788A CN 202310761031 A CN202310761031 A CN 202310761031A CN 116515788 A CN116515788 A CN 116515788A
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- 230000003197 catalytic effect Effects 0.000 claims description 57
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Classifications
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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
- 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
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- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- 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)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The present invention provides novel R-type ω -aminotransferases comprising at least one of ω -aminotransferase TA-R1, ω -aminotransferase TA-R2 or ω -aminotransferase TA-R3, the amino acid sequence of ω -aminotransferase TA-R1 being as set forth in Seq ID NO:1, and the amino acid sequence of the omega-aminotransferase TA-R2 is shown as Seq ID NO:2, the amino acid sequence of the omega-aminotransferase TA-R3 is as shown in Seq ID NO: 3. The R-type omega-aminotransferase TA-R1, TA-R2 and TA-R3 and genes thereof solve the problem of relatively insufficient R-type omega-aminotransferase resources in the existing chiral amine asymmetric biosynthesis technology, and develop a novel R-type chiral amine compound asymmetric biosynthesis method based on new enzyme resources.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to R-type omega-aminotransferase, and a gene and application thereof.
Background
Chiral amine drugs are an important class of chiral drugs, and many drug molecules have chiral amine structural modules in the synthesis process. For example, the hypoglycemic drugs sitagliptin, the antibacterial drugs moxifloxacin and the like all contain chiral amine structural units. The development of economic, green, specific and efficient chiral amine synthesis technology is an important aspect in the field of chiral drug synthesis and development. At present, chiral amine compounds can be synthesized in three modes, namely chemical synthesis, kinetic resolution and asymmetric biocatalysis synthesis. The traditional chemical synthesis method generally has the advantages of clear reaction system and good universality, but also has the problems of more reaction steps, limited stereoselectivity, heavy metal pollution, high catalyst cost and the like. The dynamic resolution method has the advantage of simple reaction, and the racemic amine is subjected to chiral resolution by using a proper catalyst, but the highest theoretical conversion rate of the method is 50%, so that the industrial application potential of the method is greatly limited. In contrast, the asymmetric catalysis synthesis of chiral amine by using omega-aminotransferase has the characteristics of simple reaction, mild process, high stereospecificity and the like, and has great application potential in chiral amine synthesis production. In 2010, researchers of Merck company and Codexis company apply protein engineering technology to modify and obtain omega-aminotransferase mutant ATA117 with strong solvent tolerance and high catalytic activity, and the omega-aminotransferase mutant ATA117 is successfully used in the catalytic synthesis process of the hypoglycemic drug sitagliptin, so that the great simplification of the sitagliptin production process and the remarkable improvement of the production efficiency are realized (2010 Science 329:305-309).
Obtaining omega-aminotransferase with the specificity of the stereo catalysis is a precondition for asymmetric biosynthesis of chiral amines. Because the abundance of R-type ω -transaminase is far lower than that of S-type in nature, the currently available R-type ω -transaminase catalyst resources are very limited, which limits the synthesis of R-type chiral amine compounds. In order to make up the current situation that R-type omega-aminotransferase is relatively insufficient in resources and develop a novel R-type chiral amine compound synthesis method, the invention utilizes an enzyme element excavation research technology driven by a machine learning algorithm to excavate and obtain a novel R-type omega-aminotransferase from an ocean metagenome database, and the novel R-type omega-aminotransferase is used for biosynthesis of the R-type chiral amine compound.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides novel R-type omega-aminotransferase and a gene thereof, solves the problem that R-type omega-aminotransferase resources are relatively insufficient in the existing chiral amine asymmetric biosynthesis technology, and develops a novel R-type chiral amine compound asymmetric biosynthesis method based on new enzyme resources.
The present invention provides novel R-type ω -aminotransferases comprising at least one of ω -aminotransferase TA-R1, ω -aminotransferase TA-R2 or ω -aminotransferase TA-R3, the amino acid sequence of ω -aminotransferase TA-R1 being as set forth in Seq ID NO:1, and the amino acid sequence of the omega-aminotransferase TA-R2 is shown as Seq ID NO:2, the amino acid sequence of the omega-aminotransferase TA-R3 is as shown in Seq ID NO: 3.
The invention also provides the gene of R-type omega-aminotransferase, the gene sequence comprises at least one of the following (1), (2) and (3),
(1) the R-type omega-aminotransferase is omega-aminotransferase TA-R1, and the omega-aminotransferase TA-R1 gene sequence is a nucleotide sequence shown in the following a) or b): a) Such as Seq ID NO:4, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:4, such that a nucleotide sequence as set forth in Seq ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1;
(2) the R-type omega-aminotransferase is omega-aminotransferase TA-R2, and the gene sequence is a nucleotide sequence shown in the following c) or d): c) Such as Seq ID NO:5, a nucleotide sequence shown in seq id no; d) Such as Seq ID NO:5, such that a nucleotide sequence as set forth in Seq ID NO:2, and a nucleotide sequence of the amino acid sequence shown in seq id no;
(3) the R-type omega-aminotransferase is omega-aminotransferase TA-R3, and the omega-aminotransferase TA-R3 gene sequence is a nucleotide sequence shown in the following e) or f): e) Such as Seq ID NO:6, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:6, such that a nucleotide sequence as set forth in Seq ID NO:3, and a nucleotide sequence of the amino acid sequence shown in 3.
Preferably, the novel R-type ω -transaminase TA-R1 provided by the present invention comprises, for example, the amino acid sequence of Seq ID NO:1, and a polypeptide having the amino acid sequence shown in 1.
The invention utilizes the disclosed marine microorganism metagenome data resource OM-RGC (2015 Science 348:1261359) as a data source to excavate R-type omega-aminotransferase resources. Firstly, constructing a BLOSUM matrix according to an R-type omega-aminotransferase protein sequence with known functions, establishing a corresponding hidden Markov model according to analysis of the matrix, and searching in an OM-RGC database by using the model to obtain the novel R-omega-aminotransferase TA-R1, wherein the amino acid sequence of the novel R-omega-aminotransferase TA-R1 is as shown in Seq ID NO:1, the protein size is 309 amino acid residues. In contrast to a typical R-type ω -transaminase (Genebank accession BAK 39753.1) from Arthrobacter sp, TA-R1 has 33% sequence identity thereto; TA-R1 also showed only 37% sequence identity compared to another typical R-type ω -transaminase (Genebank accession XP_ 001209325.1) derived from A.terreus (Aspergillus terreus).
Preferably, the R-type omega-aminotransferase TA-R1 has aminotransferase activity for at least one amino acceptor selected from benzaldehyde, acetophenone, furan-2-formaldehyde, cyclohexane ketone, hexanal, 2-hexanone and sodium pyruvate. Preferably, any of the above-mentioned materials is used, wherein the catalytic temperature of the R-type omega-aminotransferase TA-R1 is 25-45 ℃. Further preferred catalytic temperatures are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃. Preferably, any of the above-mentioned compounds is a type R ω -transaminase TA-R1, the pH of the catalytic system is from 6.5 to 9.0. Further preferred catalytic systems have a pH of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0.
Preferably, any of the above-mentioned amino donor/acceptor molar ratios in the catalytic system of the ω -transaminase type R TA-R1: 1:1-20:1. Further preferably, 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1. 10: 1. 11: 1. 12: 1. 13: 1. 14: 1. 15: 1. 16: 1. 17: 1. 18: 1. 19: 1. 20:1.
preferably, any of the above-mentioned R-type ω -transaminase TA-R1 catalytic system comprises pyridoxal phosphate (PLP) in an amount of 0.1-2.0 mM. More preferably, 0.1, 0.3, 0.7, 1.0, 1.4, 1.8, 2.0mM.
Preferably, any one of the above-mentioned materials contains 5% -20% DMSO (volume percentage) in the R-type omega-aminotransferase TA-R1 catalytic system. More preferably, 5%, 8%, 10%, 12%, 15%, 17%, 20%,10% to 20%.
Preferably, any of the above-mentioned catalyst systems for the ω -transaminase type R TA-R1 comprise a phosphate buffer solution or a triethanolamine buffer solution in a concentration ranging from 0.02 to 0.15M. More preferably, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15M.
Preferably, any one of the above-mentioned catalytic systems for ω -transaminase type R TA-R1 comprise: 0.1M potassium phosphate buffer, 10 mM amino acceptor, 5% -20% DMSO, 0.5 mM pyridoxal phosphate, 25 mM amino donor, 0.4-2.0 mg/ml R-type omega-aminotransferase TA-R1.
Preferably, any of the above amino donors comprises 4-nitrophenethylamine, R-methylbenzylamine.
In a preferred embodiment of the invention the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM amino acceptors, 20% dimethyl sulfoxide (DMSO), 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4-2.0 mg/ml aminotransferase TA-R1 catalyst, and at 30℃for 18 hours with shaking at 500rpm, TA-R1 has transaminase activity on at least one amino acceptor of benzaldehyde, acetophenone, furan-2-formaldehyde, cyclohexanone, hexanal, 2-hexanone, sodium pyruvate.
In a preferred embodiment of the invention, the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4 mg/ml transaminase catalyst, and is reacted for 18 hours with shaking at 400 rpm at 30℃with TA-R1 having catalytic activity on R-MBA as an amino donor and no significant catalytic activity on S-MBA.
The invention also provides a novel R-type omega-aminotransferase TA-R1 gene, the gene sequence of which is a nucleotide sequence shown in the following a) or b): a) Such as Seq ID NO:4, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:4, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1.
Preferably, the nucleotide sequence shown in b) is Seq ID No:4 is obtained by codon optimization of escherichia coli expression. The method of codon optimization is software or methods known in the art.
Preferably, the Seq ID NO:4, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID NO:1 is preferably the nucleotide sequence of the amino acid sequence shown in Seq ID NO: 7.
The invention also provides a recombinant plasmid which is constructed by connecting the gene sequence with an expression vector plasmid.
Preferably, the expression vector includes pET series, pET-GST, pGEX series (containing GST tag), pMAL series, etc., more preferably pET-28b (+) vector, pET15b, pET28a, pGEX4T1, pGEX-6p-1, etc. Preferably, the Seq ID NO:4 is constructed on a pET-28b (+) vector to obtain a pET-28b (+) -TA-R1 plasmid.
Preferably, any one of the above-mentioned recombinant plasmid is transformed into host bacteria to obtain the TA-R1 recombinant genetic engineering strain. Preferably, the genetically engineered bacterium is escherichia coli BL21 (DE 3) transfected with pET-28b (+) -TA-R1 plasmid.
The invention also provides the R-type omega-aminotransferase TA-R1 and the application of the recombinant plasmid in the asymmetric biosynthesis of the R-type chiral amine compound.
The invention also provides the application of the recombinant engineering bacteria containing the R-type omega-aminotransferase or the recombinant plasmid and the crushing liquid thereof in the asymmetric biosynthesis of the R-type chiral amine compound.
Preferably, the invention provides a novel R-type omega-aminotransferase TA-R2, comprising, for example, seq ID NO:2, and a polypeptide having the amino acid sequence shown in 2.
The invention utilizes the disclosed marine microorganism metagenome data resource OM-RGC (2015 Science 348:1261359) as a data source to excavate R-type omega-aminotransferase resources. Firstly, constructing a BLOSUM matrix according to an R-type omega-aminotransferase protein sequence with known functions, establishing a corresponding hidden Markov model according to analysis of the matrix, and searching in an OM-RGC database by using the model to obtain the novel R-omega-aminotransferase TA-R2, wherein the amino acid sequence of the novel R-omega-aminotransferase TA-R2 is as shown in Seq ID NO:2, the protein size is 300 amino acid residues. In comparison to a typical R-type ω -transaminase (Genebank accession BAK 39753.1) from Arthrobacter sp, TA-R2 has 37% sequence identity thereto; TA-R2 also showed only 40% sequence identity compared to another typical R-type ω -transaminase (Genebank accession XP_ 001209325.1) derived from A.terreus (Aspergillus terreus).
Preferably, the R-type omega-aminotransferase TA-R2 has aminotransferase activity on sodium amino acceptor pyruvate.
Preferably, any of the above-mentioned materials is used, wherein the catalytic temperature of the R-type omega-aminotransferase TA-R2 is 25-45 ℃. Further preferred catalytic temperatures are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃.
Preferably, any of the above-mentioned compounds is a catalyst system comprising said R-type ω -transaminase TA-R2 having a pH of from 6.5 to 9.0. Further preferred catalytic systems have a pH of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0.
Preferably, any of the above-mentioned amino donor/acceptor molar ratios in the catalytic system of the ω -transaminase type R TA-R2: 1:1-20:1. Further preferably, 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1. 10: 1. 11: 1. 12: 1. 13: 1. 14: 1. 15: 1. 16: 1. 17: 1. 18: 1. 19: 1. 20:1.
preferably, any of the above-mentioned R-type ω -transaminase TA-R2 catalytic system comprises pyridoxal phosphate (PLP) in an amount of 0.1-2.0 mM. More preferably, 0.1, 0.3, 0.7, 1.0, 1.4, 1.8, 2.0mM.
Preferably, any one of the above-mentioned materials contains 5% -20% DMSO (volume percentage) in the R-type omega-aminotransferase TA-R2 catalytic system. More preferably, 5%, 8%, 10%, 12%, 15%, 17%, 20%,10% to 20%.
Preferably, any of the above-mentioned catalyst systems for the ω -transaminase type R TA-R2 comprise a phosphate buffer solution or a triethanolamine buffer solution in a concentration ranging from 0.02 to 0.15M. More preferably, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15M.
Preferably, any one of the above-mentioned catalyst systems comprises: 0.1M potassium phosphate buffer, 10 mM amino acceptor sodium pyruvate, 5 to 20 percent DMSO, 0.5 to mM pyridoxal phosphate, 25 mM amino donor and 0.4 to 2.0 mg/ml R-type omega-aminotransferase TA-R2.
Preferably, any of the above amino donors comprises 4-nitrophenethylamine, R-methylbenzylamine.
In a preferred embodiment of the invention the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM amino acceptor sodium pyruvate, 20% dimethyl sulfoxide (DMSO), 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4 to 2.0 mg/ml transaminase TA-R2 catalyst, and the reaction is carried out for 18 hours at 30℃with shaking at 500rpm, TA-R2 having transaminase activity towards sodium pyruvate.
In a preferred embodiment of the invention, the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10. 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4 mg/ml transaminase catalyst, and is reacted for 18 hours with shaking at 400 rpm at 30℃with TA-R2 being catalytically active for R-MBA as an amino donor and no significant catalytic activity for S-MBA.
The invention also provides a novel R-type omega-aminotransferase TA-R2 gene, the gene sequence of which is the nucleotide sequence shown in the following c) or d): c) Such as Seq ID NO:5, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:5, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID NO:2, and a nucleotide sequence of the amino acid sequence shown in seq id no.
d) The nucleotide sequence shown in c) is obtained by optimizing an escherichia coli expression codon. Methods of codon optimization are known in the art.
Preferably, the Seq ID NO:5, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID NO:2 is preferably the nucleotide sequence of the amino acid sequence shown in Seq ID NO:8, and a nucleotide sequence shown in SEQ ID NO.
The invention also provides a recombinant plasmid which is constructed by connecting the gene sequence with an expression vector plasmid.
Preferably, the expression vector includes pET series, pET-GST, pGEX series (containing GST tag), pMAL series, etc., more preferably pET-28b (+) vector, pET15b, pET28a, pGEX4T1, pGEX-6p-1, etc. Preferably, the Seq ID NO:5 is constructed on a pET-28b (+) vector to obtain a pET-28b (+) -TA-R2 plasmid.
Preferably, any one of the above-mentioned recombinant plasmid is transformed into host bacteria to obtain the TA-R2 recombinant genetic engineering strain. Preferably, the genetically engineered bacterium is escherichia coli BL21 (DE 3) transfected with pET-28b (+) -TA-R2 plasmid.
The invention also provides R-type omega-aminotransferase TA-R2 and the recombinant plasmid, engineering bacteria containing the R-type omega-aminotransferase TA-R2 and the recombinant plasmid, and application of the engineering bacteria and crushing liquid thereof in asymmetric biosynthesis of R-type chiral amine compounds.
Preferably, the R-form ω -transaminase TA-R2 has transaminase activity on the amino receptor sodium pyruvate.
Preferably, the catalytic temperature of the R-type omega-aminotransferase TA-R2 is 25-45 ℃. Further preferred catalytic temperatures are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃.
Preferably, the pH of the R-type omega-aminotransferase TA-R2 catalytic system is from 6.5 to 9.0. Further preferred catalytic systems have a pH of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0.
Preferably, any of the above-mentioned amino donor/acceptor molar ratios in the catalytic system of the ω -transaminase type R TA-R2: 1:1-20:1. Further preferably, 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1. 10: 1. 11: 1. 12: 1. 13: 1. 14: 1. 15: 1. 16: 1. 17: 1. 18: 1. 19: 1. 20:1.
preferably, any of the above-mentioned R-type ω -transaminase TA-R2 catalytic system comprises pyridoxal phosphate (PLP) in an amount of 0.1-2.0 mM. More preferably, 0.1, 0.3, 0.7, 1.0, 1.4, 1.8, 2.0mM.
Preferably, any one of the above-mentioned materials contains 5% -20% DMSO (volume percentage) in the R-type omega-aminotransferase TA-R2 catalytic system. More preferably, 5%, 8%, 10%, 12%, 15%, 17%, 20%,10% to 20%.
Preferably, any of the above-mentioned catalyst systems for the ω -transaminase type R TA-R2 comprise a phosphate buffer solution or a triethanolamine buffer solution in a concentration ranging from 0.02 to 0.15M. More preferably, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15M.
Preferably, the R-type ω -transaminase TA-R2 catalytic system comprises: 0.1M potassium phosphate buffer, 10 mM amino acceptor, 5% -20% DMSO, 0.5 mM pyridoxal phosphate, 25 mM amino donor, 0.4-2.0 mg/ml R-type omega-aminotransferase TA-R2.
Preferably, the amino donor of the R-type omega-aminotransferase TA-R2 comprises 4-nitrophenethylamine and R-type methylbenzylamine.
Preferably, the novel R-type ω -transaminase TA-R3 provided by the present invention includes, for example, seq ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
The invention utilizes the disclosed marine microorganism metagenome data resource OM-RGC (2015 Science 348:1261359) as a data source to excavate R-type omega-aminotransferase resources. Firstly, constructing a BLOSUM matrix according to an R-type omega-aminotransferase protein sequence with known functions, establishing a corresponding hidden Markov model according to analysis of the matrix, and searching in an OM-RGC database by using the model to obtain the novel R-omega-aminotransferase TA-R3, wherein the amino acid sequence of the novel R-omega-aminotransferase TA-R3 is as shown in Seq ID NO:3, the protein size is 334 amino acid residues. In comparison to a typical R-type ω -transaminase (Genebank accession BAK 39753.1) from Arthrobacter sp, TA-R3 has 35% sequence identity thereto; TA-R3 also showed only 34% sequence identity compared to another typical R-type ω -transaminase (Genebank accession XP_ 001209325.1) derived from A.terreus (Aspergillus terreus).
Preferably, the R-type omega-aminotransferase TA-R3 has aminotransferase activity on sodium amino acceptor pyruvate.
Preferably, any of the above-mentioned materials is used, wherein the catalytic temperature of the R-type omega-aminotransferase TA-R3 is 25-45 ℃. Further preferred catalytic temperatures are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 ℃.
Preferably, any of the above-mentioned compounds is a catalyst system comprising said R-type ω -transaminase TA-R3 having a pH of from 6.5 to 9.0. Further preferred catalytic systems have a pH of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0.
Preferably, any of the above-mentioned amino donor/acceptor molar ratios in the catalytic system of the ω -transaminase type R TA-R3: 1:1-20:1. Further preferably, 1: 1. 2: 1. 3: 1. 4: 1. 5: 1. 6: 1. 7: 1. 8: 1. 9: 1. 10: 1. 11: 1. 12: 1. 13: 1. 14: 1. 15: 1. 16: 1. 17: 1. 18: 1. 19: 1. 20:1.
preferably, any of the above-mentioned R-type ω -transaminase TA-R3 catalytic system comprises pyridoxal phosphate (PLP) in an amount of 0.1-2.0 mM. More preferably, 0.1, 0.3, 0.7, 1.0, 1.4, 1.8, 2.0mM.
Preferably, any one of the above-mentioned materials contains 5% -20% DMSO (volume percentage) in the R-type omega-aminotransferase TA-R3 catalytic system. More preferably, 5%, 8%, 10%, 12%, 15%, 17%, 20%,10% to 20%.
Preferably, any of the above-mentioned catalyst systems for the ω -transaminase type R TA-R3 comprise a phosphate buffer solution or a triethanolamine buffer solution in a concentration ranging from 0.02 to 0.15M. More preferably, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15M.
Preferably, any one of the above-mentioned catalytic systems comprises: 0.1M potassium phosphate buffer, 10 mM amino acceptor, 5% -20% DMSO, 0.5 mM pyridoxal phosphate, 25 mM amino donor, 0.4-2.0 mg/ml R-type omega-aminotransferase TA-R3.
Preferably, the amino acceptor is sodium pyruvate.
Preferably, any of the above amino donors comprises 4-nitrophenethylamine, R-methylbenzylamine.
In a preferred embodiment of the invention the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10 mM amino acceptors, 20% dimethyl sulfoxide (DMSO), 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4 to 2.0 mg/ml aminotransferase TA-R3 catalyst, and the TA-R3 has aminotransferase activity on sodium amino acceptors by shaking reaction at 500rpm for 18 hours at 30 ℃.
In a preferred embodiment of the invention, the catalytic system is a 0.1M potassium phosphate buffer (pH 7.5) containing 10. 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4 mg/ml transaminase catalyst, and the reaction is carried out at 30℃for 18 hours with shaking at 400 rpm, TA-R3 being catalytically active for the R-MBA as an amino donor and not significantly catalytically active for the S-MBA.
The invention also provides a novel R-type omega-aminotransferase TA-R3 gene, the gene sequence of which is a nucleotide sequence shown in the following e) or f): e) Such as Seq ID NO:6, a nucleotide sequence shown in seq id no; f) Such as Seq ID NO:6, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID No:3, and a nucleotide sequence of the amino acid sequence shown in 3. Preferably, the Seq ID NO:6, and obtaining the nucleotide sequence shown in f) through the codon optimization of the escherichia coli. The codon optimization method is a method described in the prior art.
Preferably, the Seq ID NO:6, by one or more base changes, such that it obtains a nucleotide sequence as set forth in Seq ID No:3 is preferably the nucleotide sequence of the amino acid sequence shown in Seq ID NO: 9.
The invention also provides a recombinant plasmid which is constructed by connecting the gene sequence with an expression vector plasmid.
Preferably, the expression vector includes pET series, pET-GST, pGEX series (containing GST tag), pMAL series, etc., more preferably pET-28b (+) vector, pET15b, pET28a, pGEX4T1, pGEX-6p-1, etc. Preferably, the Seq ID NO:6, constructing the TA-R3 sequence coded by the gene into a pET-28b (+) vector to obtain a pET-28b (+) -TA-R3 plasmid.
Preferably, any one of the above-mentioned recombinant plasmid is transformed into host bacteria to obtain the TA-R3 recombinant genetic engineering strain. Preferably, the genetically engineered bacterium is escherichia coli BL21 (DE 3) transfected with pET-28b (+) -TA-R3 plasmid.
The invention also provides the R-type omega-aminotransferase TA-R3 and the recombinant plasmid, or the application of the genetically engineered bacterium containing the R-type omega-aminotransferase TA-R3 or the recombinant plasmid and the crushing liquid thereof in the asymmetric biosynthesis of the R-type chiral amine compound.
Drawings
FIG. 1 is a SDS-PAGE map of TA-R1 protein expression in preferred embodiment 3 of the invention.
FIG. 2 is a SDS-PAGE map of TA-R2 protein expression in preferred embodiment 9 of the invention.
FIG. 3 is a SDS-PAGE map of TA-R3 protein expression in the preferred embodiment 15 of the invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
The foregoing examples are provided for clarity of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Examples 1 to 6 are preferred embodiments for the preparation and screening of the R-type ω -transaminase TA-R1, and confirmation and analysis of the activity and catalytic steric properties of the transaminase TA-R1.
Example 1
Construction of recombinant plasmid containing TA-R1 aminotransferase Gene
With Seq ID NO:1 as template, and performing codon optimization of DNA sequence for colibacillus to synthesize aminotransferase gene for heterologous expression, the nucleotide sequence of which is shown in SEQ ID No. 4. The NdeI and XhoI restriction sites are utilized to clone a target gene onto a pET-28b (+) vector through a molecular cloning technology, so as to obtain a recombinant plasmid for expressing TA-R1 aminotransferase, and the recombinant plasmid is transformed into an escherichia coli DH5 alpha strain through a chemical transformation method. Positive clones were obtained on LB plates containing kanamycin, single colonies were picked, cultured in LB medium, plasmids were extracted, and the sequencing result confirmed the correctness of the DNA sequence of the target aminotransferase gene.
Example 2
Construction of recombinant genetically engineered Strain containing TA-R1 aminotransferase
Mu.l of the recombinant expression plasmid obtained in example 1 was transformed into 100. Mu.l of competent cells of E.coli BL21 (DE 3) by heat shock at 42℃for 90 seconds, resuscitated at 37℃for 1 hour, and 100. Mu.l of the cell suspension was taken out and plated on LB solid medium containing 50. Mu.g/ml kanamycin. At 37℃the culture was allowed to stand overnight. And (5) selecting a single colony to verify positive cloning, and obtaining the recombinant genetically engineered bacterium.
Example 3
Inducible expression of target aminotransferase TA-R1 and preparation of aminotransferase TA-R1 catalyst
A monoclonal colony of the recombinant genetically engineered bacterium is picked up, inoculated into 10ml of LB liquid medium containing 50 mug/ml kanamycin, and cultured overnight at 37 ℃ in a shaking table. The overnight cultures were inoculated at 1% inoculum size into 500 ml LB liquid medium containing 50. Mu.g/ml kanamycin, shake-cultured at 37℃to logarithmic growth phase, added with 0.3 mM IPTG, and induced target aminotransferase expression at 20 ℃. After 20 hours of induction, 1ml of the culture was centrifuged to collect the cells, and SDS-PAGE (denaturing polyacrylamide gel electrophoresis) was used to check whether the aminotransferase was successfully expressed in the genetically engineered bacteria, as shown in FIG. 1, wherein 1 is the pre-induction expression case, 2 is the post-induction expression case, and the triangles indicate the position of the target aminotransferase in SDS-PAGE, and the apparent molecular weight thereof in the gel map is marked aside. For cells successfully expressing aminotransferase, the induced culture was centrifuged, cells were collected and resuspended in Tris-HCl buffer (ph 8.0) of 20 mM, the cells were disrupted by sonication, and the supernatant after centrifugation was used as a catalyst for the reaction.
Example 4
Preliminary screening for aminotransferase TA-R1 Activity
The preliminary screening of the aminotransferase activity was carried out in a 0.2. 0.2 ml reaction system, which was a 0.1M potassium phosphate buffer (pH 7.5), containing 10 mM amino acceptors (benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexane ketone, hexanal, 2-hexanone, sodium pyruvate), 20% dimethyl sulfoxide (DMSO), 0.5. 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4-2.0 mg/ml aminotransferase catalyst, and the reaction was carried out for 18 hours with shaking at 500rpm under 30℃conditions, and the color change of the reactants was observed, with red precipitation, representing aminotransferase activity. The results show that at least one amino acceptor of TA-R1 p-benzaldehyde, acetophenone, furan-2-formaldehyde, cyclohexane ketone, hexanal, 2-hexanone and sodium pyruvate has transaminase activity.
Example 5
Confirmation of aminotransferase TA-R1 Activity and analysis of catalytic stereospecificity
The activity of transaminase was confirmed and the analysis of the catalytic steric characteristics was carried out in a 0.2. 0.2 ml reaction system comprising 0.1M potassium phosphate buffer solution (pH 7.5) containing 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4. 0.4 mg/ml transaminase catalyst, and the reaction was terminated by adding an equal volume of methanol, and then the acetophenone product was detected by HPLC, and the target transaminase catalytic activity was evaluated based on the substrate sodium pyruvate conversion. As a result, TA-R1 had transaminase activity and substrate conversion rates of 51.3% respectively, as shown in the following Table; in addition, TA-R1 specifically utilizes R-MBA as an amino donor, and has no obvious catalytic activity on S-MBA, as shown in the following table. The above results confirm that TA-R1 is an R-type ω -transaminase.
Example 6
The nucleotide sequence shown in SEQ ID No.7 is used for constructing a recombinant plasmid of a TA-R1 aminotransferase gene and a TA-R1 recombinant gene engineering strain. The obtained TA-R1 transaminase is R-type omega-transaminase, and has transaminase activity on at least one amino receptor selected from benzaldehyde, acetophenone, furan-2-formaldehyde, cyclohexanone, hexanal, 2-hexanone and sodium pyruvate.
(II) examples 7 to 12 are preferred embodiments for the preparation and screening of the R-type ω -transaminase TA-R2, and confirmation and analysis of the activity and catalytic steric properties of the transaminase TA-R2.
Example 7
Construction of recombinant plasmid pET-28b (+) -TA-R2
Optimizing the Seq ID NO:2, and obtaining a nucleotide sequence shown in SEQ ID No. 5. The aminotransferase gene for heterologous expression shown in SEQ ID No.5, i.e., the target gene, was obtained by synthesis. Cloning the target gene to a pET-28b (+) vector by utilizing NdeI and XhoI enzyme cutting sites through a molecular cloning technology to obtain a recombinant plasmid pET-28b (+) -TA-R2 for expressing TA-R2 aminotransferase, and transforming into an escherichia coli DH5 alpha strain. Positive clone strains were obtained on LB plates containing kanamycin, single colonies were picked, and after continuous culture in LB medium, plasmids were extracted, and the sequencing results confirmed the correctness of pET-28b (+) -TA-R2.
Example 8
Construction of recombinant genetically engineered Strain containing TA-R2 aminotransferase
Mu.l of the recombinant expression plasmid pET-28b (+) -TA-R2 obtained in example 7 was transformed into 100. Mu.l of competent cells of E.coli BL21 (DE 3) by heat shock at 42℃for 90 seconds, and resuscitated at 37℃for 1 hour, 100. Mu.l of the cell suspension was taken out and spread on LB solid medium containing 50. Mu.g/ml kanamycin. At 37℃the culture was allowed to stand overnight. And (5) selecting a single colony to verify positive cloning, and obtaining the recombinant genetically engineered bacterium.
Example 9
Inducible expression of target aminotransferase TA-R2 and preparation of TA-R2 aminotransferase catalyst
A monoclonal colony of the recombinant genetically engineered bacterium is picked up, inoculated into 10ml of LB liquid medium containing 50 mug/ml kanamycin, and cultured overnight at 37 ℃ in a shaking table. The overnight cultures were inoculated at 1% inoculum size into 500 ml LB liquid medium containing 50. Mu.g/ml kanamycin, shake-cultured at 37℃to logarithmic growth phase, added with 0.3 mM IPTG, and induced target aminotransferase expression at 20 ℃. After 20 hours of induction, 1ml of the culture was centrifuged to collect the cells, and SDS-PAGE (denaturing polyacrylamide gel electrophoresis) was used to check whether the aminotransferase was successfully expressed in the genetically engineered bacteria, as shown in FIG. 2, wherein 1 is the pre-induction expression case and 2 is the post-induction expression case, and triangles indicate the position of the target aminotransferase in SDS-PAGE, and the apparent molecular weight thereof in the gel map is marked aside. For cells successfully expressing aminotransferase, the induced culture was centrifuged, cells were collected and resuspended in Tris-HCl buffer (ph 8.0) of 20 mM, the cells were disrupted by sonication, and the supernatant after centrifugation was used as a catalyst for the reaction.
Example 10
Preliminary screening for aminotransferase TA-R2 Activity
The preliminary screening of the aminotransferase activity was carried out in a 0.2. 0.2 ml reaction system, which was a 0.1M potassium phosphate buffer (pH 7.5), containing 10 mM amino acceptors (benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexane ketone, hexanal, 2-hexanone, sodium pyruvate), 20% dimethyl sulfoxide (DMSO), 0.5. 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4-2.0 mg/ml aminotransferase catalyst, and the reaction was carried out for 18 hours with shaking at 500rpm under 30℃conditions, and the color change of the reactants was observed, with red precipitation, representing aminotransferase activity. The results show that TA-R2 has transaminase activity on sodium pyruvate.
Example 11
Confirmation of aminotransferase TA-R2 Activity and analysis of catalytic stereospecificity
The activity of transaminase was confirmed and the analysis of the catalytic steric characteristics was carried out in a 0.2. 0.2 ml reaction system comprising 0.1M potassium phosphate buffer solution (pH 7.5) containing 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4. 0.4 mg/ml transaminase catalyst, and the reaction was terminated by adding an equal volume of methanol, and then the acetophenone product was detected by HPLC, and the target transaminase catalytic activity was evaluated based on the substrate sodium pyruvate conversion. As a result, TA-R2 had transaminase activity and the substrate conversion was 46.6% as shown in the following Table; in addition, TA-R2 specificity utilizes R-MBA as an amino donor, and has no obvious catalytic activity on S-MBA, as shown in the following table. The above results confirm that TA-R2 is an R-type ω -transaminase.
Example 12
The nucleotide sequence shown in SEQ ID No.8 is used for constructing a recombinant plasmid of a TA-R2 aminotransferase gene and a TA-R2 recombinant genetic engineering strain. The obtained TA-R2 transaminase is R-type omega-transaminase, and has transaminase activity on sodium pyruvate.
(III) examples 13 to 18 are preferred embodiments for the preparation and screening of the R-type ω -transaminase TA-R3, and confirmation and analysis of the activity and catalytic steric properties of the transaminase TA-R3.
Example 13
Construction of recombinant plasmid containing TA-R3 aminotransferase Gene
With Seq ID NO:3, and performing codon optimization of a DNA sequence aiming at escherichia coli to synthesize a transaminase gene for heterologous expression, wherein the nucleotide sequence of the transaminase gene is shown as SEQ ID No. 6. The NdeI and XhoI restriction sites are utilized to clone a target gene onto a pET-28b (+) vector through a molecular cloning technology, so as to obtain a recombinant plasmid for expressing TA-R3 aminotransferase, and the recombinant plasmid is transformed into an escherichia coli DH5 alpha strain through a chemical transformation method. Positive clones were obtained on LB plates containing kanamycin, single colonies were picked, cultured in LB medium, plasmids were extracted, and the sequencing result confirmed the correctness of the DNA sequence of the target aminotransferase gene.
Example 14
Construction of recombinant genetically engineered Strain containing TA-R3 aminotransferase
Mu.l of the recombinant expression plasmid obtained in example 13 was transformed into 100. Mu.l of competent cells of E.coli BL21 (DE 3) by heat shock at 42℃for 90 seconds, resuscitated at 37℃for 1 hour, and 100. Mu.l of the cell suspension was taken out and plated on LB solid medium containing 50. Mu.g/ml kanamycin. At 37℃the culture was allowed to stand overnight. And (5) selecting a single colony to verify positive cloning, and obtaining the recombinant genetically engineered bacterium.
Example 15
Inducible expression of target aminotransferase TA-R3 and preparation of TA-R3 aminotransferase catalyst
A monoclonal colony of the recombinant genetically engineered bacterium is picked up, inoculated into 10ml of LB liquid medium containing 50 mug/ml kanamycin, and cultured overnight at 37 ℃ in a shaking table. The overnight cultures were inoculated at 1% inoculum size into 500 ml LB liquid medium containing 50. Mu.g/ml kanamycin, shake-cultured at 37℃to logarithmic growth phase, added with 0.3 mM IPTG, and induced target aminotransferase expression at 20 ℃. After 20 hours of induction, 1ml of the culture was centrifuged to collect the cells, and SDS-PAGE (denaturing polyacrylamide gel electrophoresis) was used to check whether the aminotransferase was successfully expressed in the genetically engineered bacteria, as shown in FIG. 3, wherein 1 is the pre-induction expression case, 2 is the post-induction expression case, and the triangles indicate the position of the target aminotransferase in SDS-PAGE, and the apparent molecular weight thereof in the gel map is marked aside. For cells successfully expressing aminotransferase, the induced culture was centrifuged, cells were collected and resuspended in Tris-HCl buffer (ph 8.0) of 20 mM, the cells were disrupted by sonication, and the supernatant after centrifugation was used as a catalyst for the reaction.
Example 16
Preliminary screening for aminotransferase TA-R3 Activity
The preliminary screening of the aminotransferase activity was carried out in a 0.2. 0.2 ml reaction system, which was a 0.1M potassium phosphate buffer (pH 7.5), containing 10 mM amino acceptors (benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexane ketone, hexanal, 2-hexanone, sodium pyruvate), 20% dimethyl sulfoxide (DMSO), 0.5. 0.5 mM pyridoxal phosphate (PLP), 25 mM amino donor 4-Nitrophenethylamine (NEA), 0.4-2.0 mg/ml aminotransferase catalyst, and the reaction was carried out for 18 hours with shaking at 500rpm under 30℃conditions, and the color change of the reactants was observed, with red precipitation, representing aminotransferase activity. The results show that TA-R3 has transaminase activity on sodium pyruvate.
Example 17
Confirmation of aminotransferase TA-R3 Activity and analysis of catalytic stereospecificity
The activity of transaminase was confirmed and the analysis of the catalytic steric characteristics was carried out in a 0.2. 0.2 ml reaction system comprising 0.1M potassium phosphate buffer solution (pH 7.5) containing 10 mM sodium pyruvate, 10% DMSO, 0.5 mM PLP, 25 mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4. 0.4 mg/ml transaminase catalyst, and the reaction was terminated by adding an equal volume of methanol, and then the acetophenone product was detected by HPLC, and the target transaminase catalytic activity was evaluated based on the substrate sodium pyruvate conversion. As a result, TA-R3 has transaminase activity and the substrate conversion rate is 79.6% as shown in the following Table; in addition, TA-R3 specificity utilized R-MBA as an amino donor, and had no significant catalytic activity on S-MBA, as shown in the following table. The above results confirm that TA-R3 is an R-type ω -transaminase.
Example 18
The nucleotide sequence shown in SEQ ID No.9 is used for constructing a recombinant plasmid of a TA-R3 aminotransferase gene and a TA-R3 recombinant gene engineering strain. The obtained TA-R3 transaminase is R-type omega-transaminase, and has transaminase activity on sodium pyruvate.
Experiments show that according to the technical scheme disclosed by the invention, the method comprises the following steps of: 7, an R-type ω -transaminase TA-R1 obtained from the nucleotide sequence set forth in Seq ID NO:8, or the nucleotide sequence shown in Seq ID No:9 has the same technical effects as the R-type omega-aminotransferase TA-R1, the R-type omega-aminotransferase TA-R2 and the R-type omega-aminotransferase TA-R3 obtained in example 1-example 18, and is not described in detail herein.
Claims (10)
1. Novel R-type ω -aminotransferases comprising at least one of ω -aminotransferase TA-R1, ω -aminotransferase TA-R2 or ω -aminotransferase TA-R3, the amino acid sequence of ω -aminotransferase TA-R1 being as set forth in Seq ID NO:1, and the amino acid sequence of the omega-aminotransferase TA-R2 is shown as Seq ID NO:2, the amino acid sequence of the omega-aminotransferase TA-R3 is as shown in Seq ID NO: 3.
2. The R-type ω -transaminase of claim 1, wherein the ω -transaminase TA-R1 has transaminase activity towards at least one amino-acceptor selected from the group consisting of benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexanone, hexanal, 2-hexanone, and sodium pyruvate.
3. The R-type ω -transaminase of claim 1, wherein the ω -transaminase TA-R2 has a transaminase activity on the sodium amino-receptor pyruvate.
4. The R-type ω -transaminase of claim 1, wherein the ω -transaminase TA-R3 has a transaminase activity on sodium pyruvate.
5. An R-type ω -transaminase according to any one of claims 1 to 4, in which the amino donor of the R-type ω -transaminase comprises 4-nitrophenethylamine and/or R-type methylbenzylamine.
6. The R-type ω -transaminase TA-R1 according to claim 5, wherein the catalytic system comprises: 0.02-0.15M phosphate buffer or triethanolamine buffer, 5-20% DMSO, 0.1-2.0mM pyridoxal phosphate, amino donor and acceptor with a molar ratio of 1:1-20:1, 0.4-2.0 mg/mL R-type omega-aminotransferase; the pH of the catalytic system is 6.5-9.0; the catalytic temperature is 25-45 ℃.
7. The gene for R-type ω -transaminase according to any one of claims 1 to 6, characterized in that: the gene sequence comprises at least one of the following (1), (2) and (3),
(1) the R-type omega-aminotransferase is omega-aminotransferase TA-R1, and the omega-aminotransferase TA-R1 gene sequence is a nucleotide sequence shown in the following a) or b): a) Such as Seq ID NO:4, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:4, such that a nucleotide sequence as set forth in Seq ID NO:1, and a nucleotide sequence of the amino acid sequence shown in 1;
(2) the R-type omega-aminotransferase is omega-aminotransferase TA-R2, and the gene sequence is a nucleotide sequence shown in the following c) or d): c) Such as Seq ID NO:5, a nucleotide sequence shown in seq id no; d) Such as Seq ID NO:5, such that a nucleotide sequence as set forth in Seq ID NO:2, and a nucleotide sequence of the amino acid sequence shown in seq id no;
(3) the R-type omega-aminotransferase is omega-aminotransferase TA-R3, and the omega-aminotransferase TA-R3 gene sequence is a nucleotide sequence shown in the following e) or f): e) Such as Seq ID NO:6, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:6, such that a nucleotide sequence as set forth in Seq ID NO:3, and a nucleotide sequence of the amino acid sequence shown in 3.
8. A recombinant plasmid, which is constructed by connecting the gene sequence of claim 7 with an expression vector plasmid.
9. The recombinant plasmid according to claim 8, wherein the recombinant plasmid is transformed into a host strain to obtain an R-type ω -transaminase recombinant genetically engineered strain.
10. Use of the R-type ω -transaminase according to any one of claims 1 to 6 and the recombinant plasmid according to claim 8 or 9, or an engineering bacterium or disruption solution comprising the recombinant plasmid or the R-type ω -transaminase in asymmetric biosynthesis of R-type chiral amine compounds.
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