CN116200361A - Novel R-type omega-aminotransferase TA-R1 - Google Patents
Novel R-type omega-aminotransferase TA-R1 Download PDFInfo
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Abstract
The invention provides a novel R-type omega-aminotransferase TA-R1, which comprises the following components: 1, and a polypeptide having the amino acid sequence shown in 1. The R-type omega-aminotransferase TA-R1 and the gene thereof provided by the invention solve the problem that the R-type omega-aminotransferase resource is relatively insufficient in the existing chiral amine asymmetric biosynthesis technology, and develop a novel R-type chiral amine compound asymmetric biosynthesis method based on a novel enzyme resource.
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 Codes 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 (2010Science 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 a novel R-type omega-aminotransferase TA-R1 and a gene thereof, solves the problem of relatively insufficient R-type omega-aminotransferase resources 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 invention provides a novel R-type omega-aminotransferase TA-R1, which comprises the following components: 1, and a polypeptide having the amino acid sequence shown in 1.
The invention utilizes the disclosed marine microorganism metagenome data resource OM-RGC (2015Science 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, 10mM amino acceptor, 5% -20% DMSO, 0.5mM pyridoxal phosphate, 25mM 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 10mM amino acceptor, 20% dimethyl sulfoxide (DMSO), 0.5mM pyridoxal phosphate (PLP), 25mM amino donor 4-Nitrophenethylamine (NEA), 0.4-2.0 mg/ml transaminase TA-R1 catalyst, and the reaction is carried out for 18 hours with shaking at 500rpm at 30℃with at least one amino acceptor selected from among TA-R1 having transaminase activity, acetophenone, furan-2-carbaldehyde, 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 10mM sodium pyruvate, 10% DMSO, 0.5mM PLP, 25mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4mg/ml transaminase catalyst, and the reaction is carried out for 18 hours at 30℃with shaking at 400rpm, TA-R1 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-R1 gene, the gene sequence of which is a nucleotide sequence shown in the following a) or b): a) Such as Seq ID NO:2, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:2, 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:2 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:2, 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:3, and a nucleotide sequence shown in 3.
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:2 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.
Drawings
FIG. 1 is a SDS-PAGE map of TA-R1 protein expression in preferred embodiment 3 of the invention.
Detailed Description
The present invention will be more clearly and fully described by the following examples, which are intended to be illustrative of only some, but not all, of the examples. The examples are presented to aid in understanding the invention and should not be construed to limit the scope of the invention in any way.
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. 2. 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 transaminase and preparation of transaminase 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% into 500ml of LB liquid medium containing 50. Mu.g/ml kanamycin, shake-cultured at 37℃to logarithmic growth phase, and 0.3mM IPTG was added to induce 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 20mM Tris-HCl buffer (pH 8.0), and the cells were disrupted by sonication, and the supernatant after centrifugation was used as a catalyst for the reaction.
Example 4
Preliminary screening for transaminase Activity
The preliminary screening for transaminase activity was carried out in 0.2ml of a reaction system of 0.1M potassium phosphate buffer (pH 7.5) containing 10mM of amino acceptors (benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexanones, 2-hexanone, sodium pyruvate), 20% of dimethyl sulfoxide (DMSO), 0.5mM of pyridoxal phosphate (PLP), 25mM of amino donor 4-Nitrophenethylamine (NEA), 0.4 to 2.0mg/ml of transaminase catalyst, and the reaction was carried out for 18 hours with shaking at 500rpm at 30℃to observe the color change of the reactants, with red precipitate formation representing transaminase 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 transaminase Activity and analysis of catalytic stereospecificity
The activity of transaminase was confirmed and the analysis of the catalytic stereospecificity was carried out in 0.2ml of a reaction system containing 10mM sodium pyruvate, 10% DMSO, 0.5mM PLP, 25mM R-or S-methylbenzylamine (R-MBA or S-MBA), 0.4mg/ml transaminase catalyst, shaking at 400rpm at 30℃for 18 hours, adding an equal volume of methanol to terminate the reaction, detecting acetophenone product by HPLC, and evaluating the catalytic activity of target transaminase based on the conversion of sodium pyruvate as a substrate. 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.3 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.
Claims (10)
1. A novel R-type ω -transaminase TA-R1 comprising, for example, seq ID NO:1, and a polypeptide having the amino acid sequence shown in 1.
2. The R-type ω -transaminase TA-R1 of claim 1, wherein at least one amino acceptor of p-benzaldehyde, acetophenone, furan-2-carbaldehyde, cyclohexanone, hexanal, 2-hexanone, sodium pyruvate has transaminase activity.
3. The R-type ω -transaminase TA-R1 according to claim 2, having a catalytic temperature of 25 to 45 ℃.
4. The ω -transaminase of claim 3 having a pH of from 6.5 to 9.0.
5. The R-type ω -transaminase TA-R1 according to claim 4, wherein the catalytic system comprises: 0.02-0.15M phosphate buffer solution or triethanolamine buffer solution, 5-20 percent of DMSO, 0.1-2.0mM pyridoxal phosphate, amino donor and acceptor with the mol ratio of 1:1-20:1, and 0.4-2.0 mg/mL R omega-aminotransferase TA-R1.
6. The R-type ω -transaminase TA-R1 according to claim 5, wherein the amino donor comprises 4-nitrophenethylamine, R-type methylbenzylamine.
7. A novel R-type ω -transaminase TA-R1 gene, characterized in that the gene sequence is a nucleotide sequence as shown in a) or b) below: a) Such as Seq ID NO:2, a nucleotide sequence shown in seq id no; b) Such as Seq ID NO:2, 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.
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 a TA-R1 recombinant genetically engineered strain.
10. The use of the recombinant plasmid of R-type ω -transaminase TA-R1 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 TA-R1 in asymmetric biosynthesis of R-type chiral amine compounds.
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| CN106754806B (en) * | 2016-12-20 | 2020-04-14 | 尚科生物医药（上海）有限公司 | Improved transaminase and application thereof in preparation of (R) -3-aminobutanol |
| CN109609478B (en) * | 2019-01-14 | 2020-06-16 | 浙江工业大学 | α -transaminase and mutant thereof and application thereof in asymmetric synthesis of L-glufosinate-ammonium |
| CN109609476B (en) * | 2019-01-14 | 2020-06-19 | 浙江工业大学 | α -transaminase and mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium |
| CN110982801B (en) * | 2019-12-27 | 2020-10-30 | 中国科学院苏州生物医学工程技术研究所 | Transaminase mutant and construction method and application thereof |
| CN112522228B (en) * | 2020-09-21 | 2023-12-29 | 江苏海洋大学 | R-aminotransferase from pseudomonas ammoxidation and synthesis method thereof |
| CN114277008A (en) * | 2020-09-28 | 2022-04-05 | 弈柯莱生物科技(上海)股份有限公司 | (R) -selective transaminase and application thereof |
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