CN117660389A

CN117660389A - Transaminase mutant and application thereof in preparation of chiral amine compounds

Info

Publication number: CN117660389A
Application number: CN202311669715.1A
Authority: CN
Inventors: 李培清; 汤海斌; 陆安琦
Original assignee: Zhejiang Rongrui Technology Co ltd
Current assignee: Zhejiang Rongrui Technology Co ltd
Priority date: 2023-12-04
Filing date: 2023-12-04
Publication date: 2024-03-08

Abstract

The invention provides a transaminase mutant and application thereof in preparation of chiral amine compounds. The amino acid sequence of the aminotransferase mutant is obtained by mutating the amino acid sequence of wild aminotransferase shown in SEQ ID NO. 2; the mutation mode is as follows: any one of Y164F, A245T, Y164F/A245T. The invention obtains aminotransferase mutant by site-directed mutagenesis technology based on wild aminotransferase from bacillus megatherium; the transaminase mutant can asymmetrically catalyze and synthesize 9 chiral amino alcohols and chiral amines with high added values, the highest reaction conversion rate can reach 90%, and the optical purity of the product can reach more than 99.5%; and the activity of the aminotransferase mutant is obviously improved compared with that of the wild type.

Description

Transaminase mutant and application thereof in preparation of chiral amine compounds

Technical Field

The invention relates to the technical field of biology, in particular to a transaminase mutant and application thereof in preparation of chiral amine compounds.

Background

Chiral amine compounds are important molecular building blocks in drug synthesis, with approximately 40% of drug molecules containing one or more chiral amine structures (ghis liiri, d., turner, n.j. Biocatanic Approaches to the Synthesis of Enantiomerically Pure Chiral amino. Topics in Catalysis,2014,57,284-300). For example, R-2-amino-4-phenylbutane is a precursor of the antihypertensive drug dilinolol; s-2-amino-1-propanol can be used for synthesizing Levofloxacin (Levofloxacin), is a new-generation fluoroquinolone antibacterial medicament, and is widely applied clinically at present; s-2-amino-1-butanol is a key chiral intermediate for synthesizing an antitubercular drug Ethambutol (Ethambutol); r-3-amino-1-butanol can be used for synthesizing an anti-AIDS drug Dolutegravir (dolutegradvir); s-1-methoxy-2-propylamine is a key chiral intermediate for synthesizing the chloroacetamide herbicides Metolachlor (Metolachflour) and Dimethenamid (Dimethenamid).

The synthesis of chiral amines can be divided into chemical and biological catalytic processes. The traditional chemical synthesis method mainly comprises chiral reagent resolution and chiral raw material de-novo synthesis, the highest yield is 60% -70%, the chemical synthesis method has complicated steps and severe conditions, has potential safety hazards and has serious environmental pollution (US 20110275855A1; achmato wicz, M.et al., tetrahedron,2005,61 (38): 9031-9041). For example, chinese patent application publication No. CN110066223a discloses a chemical process for preparing 1-methoxy-2-propylamine: under the condition of high temperature and high pressure, a metal catalyst is adopted to reduce and aminate the 1-methoxy-2-acetone into the 1-methoxy-2-propylamine. The method has the advantages of expensive catalyst, harsh reaction conditions, low product yield and lack of optical purity data, and does not accord with the trend of green development of pesticide chemical industry, so that the method is not suitable for large-scale industrialization.

Chiral amine compounds can also be prepared by transaminase-mediated biocatalysis. Compared with the chemical method for synthesizing chiral amine substances, which usually requires expensive metal catalysts and has the problems of low enantioselectivity and the like, the transaminase method for synthesizing chiral amine has the advantages of mild reaction conditions, high optical purity of products, green and environment-friendly process and the like, and has very wide application prospect. However, the currently studied transaminase also has the problems of poor unnatural catalytic activity, low substrate loading concentration and the like, and the wide application of the transaminase in the chiral amine industrial synthesis field is severely limited.

Therefore, development of a more efficient transaminase catalyst is needed to reduce the reaction cost and improve the potential of industrial application.

Disclosure of Invention

The invention provides a transaminase mutant and application thereof in preparing chiral amine compounds, and the transaminase mutant not only has higher enzyme activity, but also can catalyze methoxy acetone, hydroxy ketone and prochiral ketone compounds at the same time, and has high catalytic activity and conversion efficiency.

Firstly, in order to obtain aminotransferase with higher activity for catalytic preparation of chiral amine, the invention constructs a aminotransferase enzyme library, and the construction steps of the enzyme library are as follows: (1) Collecting transaminase sources and related information of gene sequences through literature investigation and multi-sequence comparison; (2) Inquiring transaminase gene sequences or amino acid sequences of different sources through a gene database (https:// www.ncbi.nlm.nih.gov/genome /), obtaining a nucleotide sequence for encoding transaminase through artificial design and codon optimization, and performing total gene synthesis by utilizing a gene synthesis technology; (3) integrating the synthesized gene fragment into an expression plasmid; (4) Transforming an expression plasmid comprising a transaminase gene into an expression host cell; (5) sequencing to verify whether the recombinant bacteria are successfully constructed; (6) Successfully constructing recombinant bacteria, numbering, and preserving in a refrigerator at-80 ℃ for later use.

And secondly, enzyme production and enzyme activity measurement are carried out through shake flask fermentation, and the optimal transaminase is obtained through screening.

And thirdly, mutating the aminotransferase obtained by screening through site-directed mutagenesis to obtain single-point mutants. And then, carrying out combined mutation on the single-point mutation to obtain different combined mutants.

Finally, the invention also provides application of the aminotransferase in catalyzing ketone compounds to react to generate amine compounds.

The specific technical scheme is as follows:

the invention provides a transaminase mutant, wherein the amino acid sequence of the transaminase mutant is obtained by mutating the amino acid sequence of wild transaminase shown in SEQ ID NO. 2;

the mutation mode is as follows: any one of Y164F, A245T, Y164F/A245T.

The mutation mode specifically refers to:

Y164F: tyrosine 164 is mutated to phenylalanine;

a245T: alanine at position 245 is mutated to threonine;

Y164F/A245T: tyrosine 164 is mutated to phenylalanine and alanine 245 is mutated to threonine.

The wild aminotransferase corresponding to the aminotransferase mutant is derived from bacillus megatherium (Bacillus megaterium), the amino acid sequence of the aminotransferase mutant is shown as SEQ ID NO.2, and the nucleotide sequence of the aminotransferase mutant is shown as SEQ ID NO. 1.

SEQ ID NO.1：ATGAGCCTGACGGTGCAGAAAATTAATTGGGAGCAGGTGAAAGA ATGGGACCGCAAGTACCTGATGCGCACGTTTAGCACCCAGAACGAATACCAGCCGGTGCCGATTGAAAGCACCGAAGGAGATTACCTTATTATGCCGGACGGTACAAGACTTCTGGATTTTTTTAACCAGCTGTATTGTGTTAACCTTGGACAAAAAAATCAGAAAGTTAACGCGGCAATTAAAGAAGCACTGGATCGTTATGGTTTCGTTTGGGATACATACGCTACCGATTATAAGGCCAAAGCAGCAAAAATTATTATCGAAGATATCCTGGGCGATGAAGATTGGCCTGGTAAAGTGAGATTTGTTAGCACCGGCAGTGAGGCAGTTGAAACCGCACTGAACATTGCAAGACTGTACACCAATCGCCCTCTGGTTGTGACCCGCGAACATGATTATCATGGTTGGACAGGTGGTGCAGCCACCGTTACCCGTCTGCGTAGCTATCGTTCAGGTCTGGTGGGGGAAAACTCTGAAAGCTTTAGCGCCCAAATTCCGGGCTCTAGCTACAATTCTGCAGTTTTAATGGCACCGAGCCCGAATATGTTTCAGGATTCAGATGGCAATTTACTGAAAGATGAGAATGGTGAGTTACTGTCGGTTAAATATACCAGACGTATGATTGAAAACTACGGTCCGGAACAGGTAGCAGCAGTTATCACGGAGGTAAGTCAGGGAGCAGGCAGTGCAATGCCGCCGTACGAATATATTCCTCAGATTCGTAAAATGACAAAGGAGCTGGGTGTGCTGTGGATTAATGATGAAGTTTTAACAGGCTTTGGTCGTACCGGTAAGTGGTTTGGGTATCAGCATTATGGTGTTCAGCCGGACATTATTACAATGGGTAAGGGACTGAGCAGTAGTAGCCTGCCGGCCGGTGCAGTTCTGGTTTCTAAAGAAATTGCAGCATTTATGGATAAGCATAGATGGGAGAGTGTTAGCACCTACGCAGGTCATCCGGTTGCAATGGCCGCAGTTTGTGCAAATTTAGAAGTTATGATGGAAGAAAACTTCGTTGAGCAAGCAAAGGATAGCGGAGAGTATATTCGTAGCAAACTGGAACTGTTACAGGAAAAACATAAAAGCATAGGTAACTTTGACGGTTATGGACTGCTGTGGATTGTGGACATTGTTAATGCAAAGACAAAAACACCGTATGTGAAATTAGATCGCAATTTTACCCACGGTATGAATCCGAATCAGATTCCGACCCAGATTATTATGAAAAAAGCACTGGAGAAAGGAGTTCTGATCGGAGGAGTTATGCCGAATACCATGCGCATTGGGGCAAGCCTGAATGTTAGTCGTGGCGATATCGATAAAGCAATGGATGCACTGGATTACGCACTTGACTATCTGGAAAGTGGTGAATGGCAGGCACTGGAACTCGAG。

SEQ ID NO.2：MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQPVPIESTEGDYLI MPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSYRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSAMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQALELE。

Further, the amino acid sequence of the aminotransferase mutant is shown as SEQ ID NO. 4; alternatively, an amino acid sequence having 80%, 85%, 90%, 95%, 98% or more than 99% homology with the amino acid sequence shown in SEQ ID NO. 4.

Further, the invention provides a gene for encoding the aminotransferase mutant, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3; alternatively, a nucleotide sequence having 80%, 85%, 90%, 95%, 98% or more than 99% homology with the nucleotide sequence shown in SEQ ID NO. 3.

SEQ ID NO.3：ATGAGCCTGACGGTGCAGAAAATTAATTGGGAGCAGGTGAAAGA ATGGGACCGCAAGTACCTGATGCGCACGTTTAGCACCCAGAACGAATACCAGAGCGTGCCGATTGAAAGCACCGAAGGAGATTACCTTATTATGCCGGACGGTACAAGACTTCTGGATTTTTTTAACCAGCTGTATTGTGTTAACCTTGGACAAAAAAATCAGAAAGTTAACGCGGCAATTAAAGAAGCACTGGATCGTTATGGTTTCGTTTGGGATACATACGCTACCGATTATAAGGCCAAAGCAGCAAAAATTATTATCGAAGATATCCTGGGCGATGAAGATTGGCCTGGTAAAGTGAGATTTGTTAGCACCGGCAGTGAGGCAGTTGAAACCGCACTGAACATTGCAAGACTGTACACCAATCGCCCTCTGGTTGTGACCCGCGAACATGATTATCATGGTTGGACAGGTGGTGCAGCCACCGTTACCCGTCTGCGTAGCTTCCGTTCAGGTCTGGTGGGGGAAAACTCTGAAAGCTTTAGCGCCCAAATTCCGGGCTCTAGCTACAATTCTGCAGTTTTAATGGCACCGAGCCCGAATATGTTTCAGGATTCAGATGGCAATTTACTGAAAGATGAGAATGGTGAGTTACTGTCGGTTAAATATACCAGACGTATGATTGAAAACTACGGTCCGGAACAGGTAGCAGCAGTTATCACGGAGGTAAGTCAGGGAGCAGGCAGTACGATGCCGCCGTACGAATATATTCCTCAGATTCGTAAAATGACAAAGGAGCTGGGTGTGCTGTGGATTAATGATGAAGTTTTAACAGGCTTTGGTCGTACCGGTAAGTGGTTTGGGTATCAGCATTATGGTGTTCAGCCGGACATTATTACAATGGGTAAGGGACTGAGCAGTAGTAGCCTGCCGGCCGGTGCAGTTCTGGTTTCTAAAGAAATTGCAGCATTTATGGATAAGCATAGATGGGAGAGTGTTAGCACCTACGCAGGTCATCCGGTTGCAATGGCCGCAGTTTGTGCAAATTTAGAAGTTATGATGGAAGAAAACTTCGTTGAGCAAGCAAAGGATAGCGGAGAGTATATTCGTAGCAAACTGGAACTGTTACAGGAAAAACATAAAAGCATAGGTAACTTTGACGGTTATGGACTGCTGTGGATTGTGGACATTGTTAATGCAAAGACAAAAACACCGTATGTGAAATTAGATCGCAATTTTACCCACGGTATGAATCCGAATCAGATTCCGACCCAGATTATTATGAAAAAAGCACTGGAGAAAGGAGTTCTGATCGGAGGAGTTATGCCGAATACCATGCGCATTGGGGCAAGCCTGAATGTTAGTCGTGGCGATATCGATAAAGCAATGGATGCACTGGATTACGCACTTGACTATCTGGAAAGTGGTGAATGGCAGGCACTGGAACTCGAG。

SEQ ID NO.4：MSLTVQKINWEQVKEWDRKYLMRTFSTQNEYQSVPIESTEGDYLI MPDGTRLLDFFNQLYCVNLGQKNQKVNAAIKEALDRYGFVWDTYATDYKAKAAKIIIEDILGDEDWPGKVRFVSTGSEAVETALNIARLYTNRPLVVTREHDYHGWTGGAATVTRLRSFRSGLVGENSESFSAQIPGSSYNSAVLMAPSPNMFQDSDGNLLKDENGELLSVKYTRRMIENYGPEQVAAVITEVSQGAGSTMPPYEYIPQIRKMTKELGVLWINDEVLTGFGRTGKWFGYQHYGVQPDIITMGKGLSSSSLPAGAVLVSKEIAAFMDKHRWESVSTYAGHPVAMAAVCANLEVMMEENFVEQAKDSGEYIRSKLELLQEKHKSIGNFDGYGLLWIVDIVNAKTKTPYVKLDRNFTHGMNPNQIPTQIIMKKALEKGVLIGGVMPNTMRIGASLNVSRGDIDKAMDALDYALDYLESGEWQALELE

Further, the present invention also provides a recombinant expression vector comprising the gene according to claim 3; preferably, the recombinant expression vector comprises a pET series vector, shuttle vector, phage or viral vector; more preferably, the recombinant expression vector is pET-28a.

Wherein the pET series vector comprises pET-22b, pET-3a, pET-3d, pET-11a, pET-12a, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b, pET-21a, pET-23b, pET-24a, pET-25b, pET-26b, pET-27b, pET-28a, pET-29a, pET-30a, pET-31b, pET-32a, pET-35b, pET-38b, pET-39b, pET-40b, pET-41a, pET-41b, pET-42a, pET-43a, pET-44a and pET-49b.

Further, the invention also provides a genetically engineered bacterium, and a host cell of the genetically engineered bacterium comprises the gene; or a recombinant expression vector as described above;

preferably, the host cell is a prokaryotic cell, yeast or eukaryotic cell; more preferably, the host cell is E.coli BL21 (DE 3).

The invention also provides an enzyme preparation, which comprises the transaminase mutant.

The invention also provides the aminotransferase mutant, the genetically engineered bacterium or the application of the enzyme preparation in catalyzing methoxy acetone to generate asymmetric amino transfer reaction to prepare (S) -1-methoxy-2-propylamine.

The invention also provides the aminotransferase mutant, the genetic engineering bacteria, or the application of the enzyme preparation in preparing chiral amino alcohol or chiral amine by catalyzing the amino transfer reaction of hydroxyketone or prochiral ketone compounds,

wherein the hydroxy ketone is 1, 3-dihydroxyacetone, 1-hydroxy-2-propanone, 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, (R) -3-hydroxy-2-butanone or (S) -3-hydroxy-2-butanone; the prochiral ketone compound is acetophenone or p-methylacetophenone; the chiral amino alcohol is serinol, (S) -alaninol, (R) -2-amino-1-butanol, (S) -3-amino-1-butanol, (2R, 3S) -3-amino-2-butanol, and (2S, 3S) -3-amino-2-butanol; the chiral amine is (S) -alpha-methylbenzylamine or (S) -1- (4-methylphenyl) ethylamine.

The invention also provides a method for preparing (S) -1-methoxy-2-propylamine by catalyzing methoxy acetone, which comprises the following steps: taking 1-methoxy-2-acetone as a substrate, and carrying out a non-para-amino transfer reaction by using a catalyst in the presence of isopropylamine or salt thereof and a cofactor to obtain (S) -1-methoxy-2-propylamine;

the catalyst is wild type transaminase or immobilized enzyme thereof; alternatively, whole cells, wet cells or crude enzyme solutions of genetically engineered bacteria comprising a wild-type transaminase; alternatively, the transaminase mutant or immobilized enzyme thereof; or whole cells, wet thalli or crude enzyme liquid of the genetically engineered bacteria; alternatively, an enzyme preparation as described;

the amino acid sequence of the wild aminotransferase is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 1.

The invention also provides a method for preparing chiral amino alcohol and/or chiral amine by catalyzing hydroxyl ketone or latent chiral ketone compounds to perform ammonia transfer reaction, which comprises the following steps:

taking hydroxy ketone and/or prochiral ketone compounds as substrates, and preparing chiral amino alcohol and/or chiral amine by utilizing a catalyst to perform ammonia transfer reaction in the presence of isopropylamine or salts thereof and cofactors;

the catalyst is wild type transaminase or immobilized enzyme thereof; alternatively, whole cells, wet cells or crude enzyme solutions of genetically engineered bacteria comprising a wild-type transaminase; alternatively, the transaminase mutant or immobilized enzyme thereof; or whole cells, wet thalli or crude enzyme liquid of the genetically engineered bacteria; alternatively, the enzyme preparation;

Compared with the prior art, the invention has the following beneficial effects:

the invention obtains aminotransferase mutant by site-directed mutagenesis technology based on wild aminotransferase from bacillus megatherium; the transaminase mutant can asymmetrically catalyze and synthesize 9 chiral amino alcohols and chiral amines with high added values, the highest reaction conversion rate can reach 90%, and the optical purity of the product can reach more than 99.5%; and the transaminase mutant obtained through protein engineering is obviously improved compared with the wild type female parent, wherein the crude enzyme activity of the optimal mutant is improved by 14.2 times compared with that of the wild type female parent, and the product e.e. is more than 99.5%.

Drawings

FIG. 1 shows the protein expression electrophoretogram of the transaminase BmeTA and its mutants (Y164F, A245T, Y F/A245T);

wherein, strip M: standard proteins; strip W: a cytopenia liquid; strip S: crushing the supernatant; strip P: the resuspension was precipitated.

FIG. 2 shows the protein purification electrophoretogram of the transaminase BmeTA and its mutants (Y164F, A245T, Y F/A245T);

wherein, strip M: standard proteins; band supernatant: crushing the supernatant; band pure enzyme: protein electrophoresis diagram after Ni-250-native Buffer elution.

FIG. 3 shows the molecular structures of (S) -1-methoxy-2-propylamine, metolachlor and dithiacet.

FIG. 4 is a reaction scheme for the transaminase-catalyzed preparation of (S) -1-methoxy-2-propylamine from 1-methoxy-2-propanone.

FIG. 5 shows the variation of HPLC of the reaction solution with and without the addition of enzyme when 4-hydroxy-2-butanone is used as a substrate;

wherein 4a is 4-hydroxy-2-butanone; (S) -4b refers to (S) -3-amino-1-butanol.

FIG. 6 shows the residual enzyme activity of the wild-type transaminase WT and of the transaminase mutants Y164F, A245T, Y164F/A245T (i.e.2M) after incubation for 30min at different temperatures.

FIG. 7 shows the residual enzyme activities of the wild-type aminotransferase WT and the aminotransferase mutants Y164F, A245T, Y164F/A245T at 40℃and 45℃for different times at 50 ℃.

Wherein A is the residual enzyme activity of BmeTA, and B is the residual enzyme activity of Y164F/A245T. .

FIG. 8 shows the different conversion of the transaminase mutants Y164F/A245T at different amino donor/substrate molar ratios in example 3.

FIG. 9 shows the catalytic substrate spectra of wild-type aminotransferase and aminotransferase mutants and the enzymatic synthesis of chiral amino alcohols and chiral amines;

wherein, the substances represented by 1a-8a are respectively: 1, 3-dihydroxyacetone, 1-hydroxy-2-propanone, 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, (R) -3-hydroxy-2-butanone or (S) -3-hydroxy-2-butanone, acetophenone and methylacetophenone;

1b-8b are serinol, (S) -alaninol, (R) -2-amino-1-butanol, (S) -3-amino-1-butanol, (2R, 3S) -3-amino-2-butanol, (2S, 3S) -3-amino-2-butanol, (S) -alpha-methylbenzylamine and (S) -1- (4-methylphenyl) ethylamine, respectively.

Detailed Description

The specific embodiments of the present invention are listed only as examples of the present invention, and the present invention is not limited to the specific embodiments described below. Any equivalent modifications and substitutions of the embodiments described below will be apparent to those skilled in the art, and are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other embodiments, methods, means, apparatus and steps well known to those skilled in the art have not been described in detail in order to not obscure the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all units used in this specification are units of international standard, and the numerical values and numerical ranges appearing in the present invention are understood to include systematic errors unavoidable in industrial production.

Example 1

1. Building enzyme library information for transaminases

Collecting transaminase sources and related information of gene sequences through literature investigation and multi-sequence comparison; querying the transaminase gene sequences or amino acid sequences of different sources through a gene database (https:// www.ncbi.nlm.nih.gov/genome /), and performing total gene synthesis; specific information on the aminotransferase obtained is shown in Table 1.

Table 1 enzyme library information for transaminases

2. Construction of strains expressing wild-type transaminase

The specific sources and accession numbers of the 36 aminotransferases in the enzyme library are shown in Table 1, and the TA 1-17 aminotransferase genes in the enzyme library were constructed on pET-28a (+) from laboratory deposit and introduced into E.coli BL21 (DE 3) host. The synthesis of the TA 18-36 aminotransferase gene, codon optimization, construction of the pET-28a (+) vector and introduction into the host E.coli BL21 (DE 3) were all performed by Beijing, the biological sciences Co. The vector pET28a and the host E.coli BL21 (DE 3) used for the study were stored in the laboratory. The restriction enzyme sites when constructing the plasmid are EcoRI/BamHI and HindIII; and (3) introducing the constructed plasmid into an expression host E.coli BL21 (DE 3) strain to obtain the genetically engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -TA 1-TA 36.

3. Recombinant expression of wild-type aminotransferase

Inoculating the successfully constructed genetically engineered bacteria into LB liquid culture medium, shake culturing at 220rpm in shaking table at 37 deg.C for 2-3 hr, and culturing when the density of bacterial cells is OD ₆₀₀ When the value reached 0.8, the temperature was lowered to 18℃and IPTG was added to a final concentration of 0.5mM. The flasks were then transferred to an 18℃shaker at 220rpm for 16h. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 15min, the supernatant was discarded, the cells were collected, and then resuspended in 100mM phosphate buffer, pH8.0, the bacterial suspension was sonicated, and the precipitate was removed by centrifugation to obtain a supernatant as a crude enzyme solution.

4. Enzyme activity assay for wild-type transaminase

The enzyme activity of the transaminase is detected by using 1-methoxy-2-acetone as a substrate.

The measurement system is as follows: the total reaction system was 1mL, 200. Mu.L of 500mM isopropyl amine hydrochloride, 200. Mu.L of 100mM substrate solution, 100. Mu.L of 10mM pyridoxal phosphate (PLP) solution, and 500. Mu.L of crude enzyme solution for cell disruption of engineering bacteria, which were prepared with 100mM phosphate buffer at pH 8.0. The reaction was quenched by shaking at 40℃for 15min and 100. Mu.L of 1M NaOH was added. The reaction mixture was centrifuged at 12000rpm for 5min to remove cells and enzyme proteins. The (S) -1-methoxy-2-propylamine produced in the reaction system was measured by high performance liquid chromatography.

The liquid chromatography (HPLC) method for detecting the optical purity of the product comprises the following steps:

1) Chromatographic conditions: chromatographic column model:QS-C18,5 μm,4.6 mm. Times.250 mm. Mobile phase: 50mM sodium acetate solution, acetonitrile=7:3. Detection wavelength: 338nm. Flow rate: 0.8mL/min. Column temperature: 40 ℃.

2) Derivatizing agent: 0.03g of phthalic dicarboxaldehyde and 0.1N-acetyl-L-cysteine are weighed respectively, dissolved with 400 mu L of ethanol, 4mL of 0.1M boric acid-sodium hydroxide buffer solution (Na2B4O7.10H2O 15.25g and NaOH 0.66g are weighed, the volume is fixed to 200mL, the ultrasonic volume is dissolved, the pH=10.0) is added, and the mixture is fully dissolved by shaking and stored in a refrigerator at 4 ℃ for standby (not more than 4 days).

3) Derivatization reaction and assay: 100. Mu.L of the sample is taken, 100. Mu.L of the derivatization reagent is added, the temperature is kept at 25 ℃ for 5min after the mixture is uniformly mixed, and 20. Mu.L of sample is injected for analysis.

The conversion of each substance in the reaction process is detected by adopting gas chromatography (QC), and the specific method comprises the following steps: chromatographic column model: GT-A capillary column, detector temperature: 240 ℃, injector temperature: 240 ℃, column furnace temperature: 70 ℃.

Definition of enzyme activity: the amount of enzyme capable of converting the substrate to 1. Mu. Mol of product at 40℃for 1min was 1U. The activity of all recombinant ω -transaminases was determined and the results are shown in table 2.

As can be seen from Table 2, the transaminase activity from Bacillus Bacillus megaterium was highest, one to two orders of magnitude higher than that of the other enzymes.

TABLE 2 transaminase enzyme activity and optical selectivity assays

Note that: NA represents undetected catalytic activity; e% means the enantiomeric excess, meaning the percentage of one isomer in the enantiomeric mixture that is greater than the other isomer.

EXAMPLE 2 construction of genetically engineered bacteria expressing mutant transaminase and Activity measurement

1. Obtaining the transaminase template Gene

Glycerol tubes storing recombinant E.coli BL21 (DE 3)/pET-28 a (+) -TA36 were streaked onto plates containing LB solid medium (50. Mu.g/mL kanamycin) and incubated at 37℃for 12h. Single colonies were picked from the plates and inoculated into 5mL of LB medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃for 12 hours at 200 rpm. After the culture broth was obtained, plasmid extraction was performed according to the instructions of the plasmid extraction kit.

2. Site-directed mutant transaminase genes

The pET-28a (+) -TA36 plasmid extracted in the step one of the embodiment 2 is used as a template, and site-directed mutation is introduced into the TA36 gene by whole plasmid PCR, and the used primers and a PCR reaction system are shown in Table 3 and Table 4 respectively.

TABLE 3 primers for PCR

TABLE 4 PCR amplification System

Component (A)	Volume (mu L)
		PrimeSTAR	20
Upstream primer	0.8
		Downstream primer	0.8
Plasmid template	0.4
		ddH ₂ O	18

Point mutation PCR was performed using the pET-28a (+) -TA36 plasmid as a template and the primers shown in Table 3, and the PCR amplification system was as shown in Table 4, and the PCR amplification conditions were as follows:

1) Pre-denaturation: 98 ℃ for 5min;

2) Denaturation: 98 ℃ for 10s; annealing: 15s at 60 ℃; extension: 1min at 72℃for 30s; cycling for 35 times;

3) Extension: 72 ℃ for 10min;

4) Preserving at 4 ℃ for 2.0h.

After the PCR amplification, the amplified product was detected by 1.0% agarose gel electrophoresis, and the target band was purified and recovered by using a DNA recovery and purification kit. The recovered PCR product was digested with DPN I and the template was removed. The digestion system is shown in Table 5.

TABLE 5 digestive system

Reagent(s)	Volume (mu L)
		PCR amplified products/plasmids	8.5
DPNI	0.5
		10×Buffer	1

Digestion conditions:

1)37℃：1h；

2)75℃：15min；

3) Preserving at 4 ℃ for 2.0h.

3. Construction of strains expressing mutant aminotransferase

After digestion in the second step of this example, the resultant was transduced into competent cells of E.coli BL21 (DE 3), and then sequenced by Beijing qingke biotechnology Co., ltd, and the plasmid with the correct sequencing result was expressed to obtain recombinant strain.

And finally obtaining 14 mutant aminotransferase mutants after induced expression.

The specific steps of the induced expression are as follows:

The 14 mutant aminotransferase mutants obtained finally were: TA36-Y164F, TA-A245T, TA36-Y164L, TA36-Y164M, TA-A245N, TA-36-A242M, TA-Y148R, TA-Y164F/A245T, TA-Y164L/A245T, TA-Y164M/A245T, TA-Y164F/A245N, TA-Y164F/A242M, TA-Y148R/A245T, TA-Y148R/A242M; the wild type was designated TA36-WT.

4. Enzyme Activity of transaminase mutants

The enzyme activity of the transaminase mutant is detected by using 1-methoxy-2-acetone as a substrate.

TABLE 6 enzyme activity measurement results of TA36 transaminase wild-type and mutant

In table 6, each mutant shown in the first column represents a sequence set forth in SEQ ID NO:2, and a specific mutation is generated in the corresponding amino acid position in the amino acid sequence shown in the formula 2. For example, Y164F depicted in Table 6 represents the sequence set forth in SEQ ID NO:2, and the 164 th tyrosine in the amino acid sequence shown in the formula 2 is mutated into phenylalanine. In the vitality column, a plus sign "+" indicates that mutant proteins are improved by 1-2 times compared with wild-type vitality; two plus signs "++" indicate that the mutant has 2-3 times higher activity than the wild type; three plus signs "++ + +" indicate that the mutant has 3-4 fold improvement over the wild-type vigor, thirteen plus signs "+++ +". As used herein, "a" + + + + + + + + + + ". The mutant has 13-14 times higher activity than wild type.

EXAMPLE 3 catalytic synthesis of (S) -1-methoxy-2-propylamine by transaminase wild-type and mutant

The transaminase-containing mutant (TA 36-Y164F, TA-A245T, TA-Y164F/A245T) with remarkably improved enzyme activity obtained in example 2 and the transaminase wild type were induced and expressed in E.coli genetically engineered bacteria to obtain a crude enzyme solution.

The specific contents are as follows: e.coli genetic engineering bacteria of the constructed transaminase wild type and transaminase mutant TA36-Y164F, TA-A245T, TA-Y164F/A245T are inoculated in LB liquid culture medium, shake culture is carried out for 2-3 h at 220rpm in a shaking table at 37 ℃, and when the density of the thallus OD is ₆₀₀ When the value reached 0.8, the temperature was lowered to 18℃and IPTG was added to a final concentration of 0.5mM. The flasks were then transferred to an 18℃shaker at 220rpm for 16h. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 15min, the supernatant was discarded, and the cells were collected, followed by 100mM phosphate pH8.0And (3) re-suspending the buffer solution, ultrasonically crushing the bacterial suspension, centrifuging to remove sediment, and obtaining a supernatant which is crude enzyme solution.

Taking crude enzyme liquid as a catalyst, catalyzing methoxy acetone to generate a non-para-amino transfer reaction to obtain (S) -1-methoxy-2-propylamine; the specific contents are as follows:

the reaction was 25mL, and the reaction conditions were 20g/L of enzyme addition, 0.5mM PLP addition, and an amino donor/amino acceptor ratio of 1.4 (700 mM isopropylamine and 500mM methoxy acetone). The reaction temperature was controlled to 30℃by a metal bath, the pH was controlled to 8.0 by 100mM phosphate buffer during the reaction, and the reaction was continued for 18 hours.

The yield of the (S) -1-methoxy-2-propylamine is 450mM, the e.e% is more than 99.5%, and the conversion rate of the raw materials reaches 90%.

By adopting the method, the embodiment also uses other transaminase mutants as catalysts to carry out non-para-aminotransferase transfer reaction to obtain (S) -1-methoxy-2-propylamine; the results are shown in Table 7.

TABLE 7 experimental results of the catalytic acquisition of (S) -1-methoxy-2-propylamine by different transaminase mutants

EXAMPLE 4 catalytic Synthesis of hydroxyketone and prochiral Ketone Compounds by transaminase mutants

The transaminase mutant (TA 36-Y164F/A245T) with remarkably improved enzyme activity obtained in example 2 is induced to express by Escherichia coli genetic engineering bacteria of transaminase wild type TA36, and crude enzyme solution is obtained.

The specific contents are as follows: e.coli genetic engineering bacteria of the constructed transaminase wild type and transaminase mutant TA36-Y164F, TA-A245T, TA-Y164F/A245T are inoculated in LB liquid culture medium, shake culture is carried out for 2-3 h at 220rpm in a shaking table at 37 ℃, and when the density of the thallus OD is ₆₀₀ The value reachesAt 0.8, the temperature was lowered to 18℃and IPTG was added to a final concentration of 0.5mM. The flasks were then transferred to an 18℃shaker at 220rpm for 16h. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 15min, the supernatant was discarded, the cells were collected, and then resuspended in 100mM phosphate buffer, pH8.0, the bacterial suspension was sonicated, and the precipitate was removed by centrifugation to obtain a supernatant as a crude enzyme solution.

Taking crude enzyme liquid as a catalyst, and taking hydroxyketone or a latent chiral ketone compound as a substrate, and carrying out ammonia transfer reaction to obtain chiral amino alcohol or chiral amine compound; the specific contents are as follows:

1mL of a reaction system was prepared under conditions of 500. Mu.L of crude enzyme solution, 100. Mu.L of 10mM PLP, 200. Mu.L of 500mM isopropylamine, and 200. Mu.L of 100mM substrate. The reaction temperature was controlled to 40℃by a metal bath, the pH was controlled to 8.0 by 100mM phosphate buffer during the reaction, and the reaction was carried out for 1 hour. Specific activity, conversion and product e.e% were measured by liquid chromatography (see in particular table 8).

TABLE 8 specific activity, conversion and e.e% results of wild-type transaminase and transaminase mutants on different substrates

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Claims

1. The amino acid sequence of the aminotransferase mutant is obtained by mutating the amino acid sequence of a wild aminotransferase shown as SEQ ID NO. 2;

the mutation mode is as follows: any one of Y164F, A245T, Y164F/A245T.

2. The transaminase mutant of claim 1, wherein the amino acid sequence of the transaminase mutant is shown in SEQ ID No. 4; alternatively, an amino acid sequence having 80%, 85%, 90%, 95%, 98% or more than 99% homology with the amino acid sequence shown in SEQ ID NO. 4. .

3. A gene encoding the transaminase mutant of claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 3; alternatively, a nucleotide sequence having 80%, 85%, 90%, 95%, 98% or more than 99% homology with the nucleotide sequence shown in SEQ ID NO. 3.

4. A recombinant expression vector comprising the gene of claim 3; preferably, the recombinant expression vector comprises a pET series vector, shuttle vector, phage or viral vector; more preferably, the recombinant expression vector is pET-28a.

5. A genetically engineered bacterium, wherein the host cell of the genetically engineered bacterium comprises the gene of claim 3; or comprising the recombinant expression vector of claim 4;

6. An enzyme preparation comprising the transaminase mutant of claim 1.

7. The use of the transaminase mutant according to any one of claims 1 or 2, or the genetically engineered bacterium according to claim 5, or the enzyme preparation according to claim 6 for catalyzing the asymmetric transamination of methoxyacetone to produce (S) -1-methoxy-2-propylamine.

8. The transaminase mutant according to any one of claims 1 or 2, or the genetically engineered bacterium according to claim 6, or the use of the enzyme preparation according to claim 7 for catalyzing the transamination of a hydroxyketone or a prochiral ketone compound to prepare a chiral amino alcohol or a chiral amine, wherein the hydroxyketone is 1, 3-dihydroxyacetone, 1-hydroxy-2-propanone, 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, (R) -3-hydroxy-2-butanone or (S) -3-hydroxy-2-butanone; the prochiral ketone compound is acetophenone or p-methylacetophenone; the chiral amino alcohol is serinol, (S) -alaninol, (R) -2-amino-1-butanol, (S) -3-amino-1-butanol, (2R, 3S) -3-amino-2-butanol, and (2S, 3S) -3-amino-2-butanol; the chiral amine is (S) -alpha-methylbenzylamine or (S) -1- (4-methylphenyl) ethylamine.

9. A method for preparing (S) -1-methoxy-2-propylamine by catalyzing methoxy acetone, which is characterized by comprising the following steps: taking 1-methoxy-2-acetone as a substrate, and carrying out a non-para-amino transfer reaction by using a catalyst in the presence of isopropylamine or salt thereof and a cofactor to obtain (S) -1-methoxy-2-propylamine;

the catalyst is wild type transaminase or immobilized enzyme thereof; alternatively, whole cells, wet cells or crude enzyme solutions of genetically engineered bacteria comprising a wild-type transaminase; alternatively, the transaminase mutant or immobilized enzyme thereof of any one of claims 1 or 2; alternatively, the genetically engineered bacterium of claim 5 is whole cells, wet cells or crude enzyme solution; alternatively, the enzyme preparation according to claim 6;

10. A method for preparing chiral amino alcohol and/or chiral amine by catalyzing hydroxyl ketone and/or latent chiral ketone compounds to generate ammonia transfer reaction, which is characterized by comprising the following steps: