CN109370998B

CN109370998B - Omega-transaminase mutant I215F with improved catalytic efficiency

Info

Publication number: CN109370998B
Application number: CN201811449478.7A
Authority: CN
Inventors: 廖祥儒; 翟李欣; 赖英杰; 杨邵岚; 蔡宇杰; 管政兵
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-08-04
Anticipated expiration: 2038-11-30
Also published as: CN109370998A

Abstract

The invention discloses a omega-transaminase mutant I215F with improved catalytic efficiency, and belongs to the technical field of genetic engineering and enzyme engineering. The invention obtains the omega-transaminase mutant with improved catalytic efficiency by carrying out site-directed mutagenesis on amino acid with higher factor B in the crystal structure of the omega-transaminase, namely mutating I at the 215 position of the omega-transaminase into F. The catalytic efficiency Kcat/Km of the mutant I215F is improved by 1.92 times, and the maximum conversion rate of acetopenone of the mutant is improved by 3.79 times compared with that of natural enzyme when the mutant catalyzes (R) -phenylenethylamine to generate the acetopenone. The mutant obtained by the invention is more suitable for industrial application than natural omega-transaminase.

Description

Omega-transaminase mutant I215F with improved catalytic efficiency

Technical Field

The invention relates to a omega-transaminase mutant I215F with improved catalytic efficiency, and belongs to the technical field of genetic engineering and enzyme engineering.

Background

Transaminase (transaminase) belongs to transferase and is generally used for catalyzing amino group to be transferred from an amino donor compound to an amino acceptor compound, protein sequences of transaminase from different sources reported in the literature are compared and clustered according to differences of different variable regions, and then the transaminase is divided into 4 types according to superposition and iterative comparison of hydrophilic sites, wherein omega-transaminase belongs to a second subfamily and is generally used for preparing chiral amine and unnatural amino acid, such as β -amino acid.

In protein molecular engineering, methods commonly used at present are rational design, irrational design and semi-rational design. The main differences between the three methods are whether the molecular structure of the enzyme protein is well understood and whether calculations and predictions need to be made using bioinformatics software. The rational design has the advantages of low experimental cost, simplicity, convenience, short time and the like.

Although the omega-transaminase has great application and research value, the catalytic efficiency of the omega-transaminase screened from wild bacteria is low, and the development and application of the omega-transaminase are greatly reduced.

Disclosure of Invention

In order to solve the problems, the invention carries out heterologous expression and site-directed mutation modification on omega-transaminase (the omega-transaminase from Bacillus pumilus W3 and the application of the omega-transaminase in biological amination 201811219769.7) from Bacillus pumilus W3, improves the catalytic rate of the omega-transaminase on specific substrates, and has profound technical guidance significance on large-scale production and popularization of the omega-transaminase.

Compared with the parent omega-transaminase, the omega-transaminase mutant has better catalytic efficiency. The parent gene is consistent with a Bacillus pumilus (Bacillus pumilus W3) omega-transaminase gene (Sequence ID: MH196528), and the plasmid template used for mutation is a carrier ota3/pCold II (application number: CN201811219769.7) carrying a natural omega-transaminase coding gene.

It is a first object of the present invention to provide a ω -transaminase mutant with improved catalytic efficiency, the amino acid sequence of the mutant comprising: the amino acid sequence obtained by mutating isoleucine at position 215 to phenylalanine on the basis of the amino acid sequence of SEQ ID NO. 1 was named I215F.

In one embodiment, the amino acid sequence of the ω -transaminase mutant is the sequence shown in SEQ ID NO. 5.

In one embodiment, the nucleotide sequence of the ω -transaminase mutant comprises the sequence shown in SEQ ID NO. 2.

A second object of the present invention is a method for preparing said mutant, comprising the steps of:

(1) designing primers for site-directed mutagenesis, carrying out mutagenesis by taking a vector carrying a omega-transaminase coding gene as a template, and constructing a plasmid vector of an I215F mutant;

(2) and (3) transforming the recombinant plasmid with the correct sequence into escherichia coli B L21 (DE3) to obtain a recombinant bacterium, fermenting and culturing the recombinant bacterium, and obtaining fermentation supernatant fluid containing the omega-transaminase mutant.

In one embodiment, the fermentation is by culturing the recombinant bacteria to OD at 37 ℃₆₀₀After that, the temperature was decreased to 15 ℃ and IPTG was added to a final concentration of 0.4mM for induction, and the mixture was centrifuged to obtain a supernatant enzyme solution after 24 hours of culture.

In one embodiment, the preparation method further comprises purifying the ω -transaminase in the fermentation supernatant using an AKTA protein purifier and a histrappf fraction 1ml nickel column.

The third purpose of the invention is to provide a recombinant plasmid vector containing the amino acid sequence of the mutant.

In one embodiment, the plasmid vector is any one of pET series, pGEX series, pCold series, or pUB.

The invention also claims a gene for coding the mutant and a gene engineering bacterium for expressing the mutant.

The invention also claims the application of the mutant, the gene for coding the mutant and the gene engineering bacteria for expressing the mutant in the catalytic synthesis of related chiral amine in the aspects of food, chemical engineering or medicament preparation, in particular the application in the preparation of medicaments.

In one embodiment, the use comprises catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound.

The applications include the selective catalysis and chiral synthesis of chiral amines, such as R-phenylethylamine, and a variety of unnatural amino acids.

The invention has the beneficial effects that:

the omega-transaminase mutant is mutated on the basis of R type omega-transaminase derived from Bacillus pumilus, and the constructed omega-transaminase mutant I215F has the performance of improving the catalytic efficiency. Enzyme kinetic analysis showed that K of mutant I215F of the invention_mThe value is reduced by 43.4 percent compared with the parent natural enzyme; catalytic efficiency K_cat/K_mThe improvement is 1.92 times. When (R) -phenylethynamine is catalyzed to generate acetophenone, the maximum conversion rate of the acetophenone of the mutant is improved by 3.79 times compared with that of parent natural enzyme. Therefore, the omega-aminotransferase mutant is more suitable for the application of the omega-aminotransferase in the process of catalyzing chiral amines such as (R) -phenylethylamine and the like than the parent.

Drawings

FIG. 1: a three-dimensional simulation structure of natural omega-transaminase;

FIG. 2: performing SDS-PAGE gel electrophoresis on the natural omega-transaminase and the mutant pure enzyme; wherein, lane 1 represents the protein molecular weight standard, lane 2 is mutant I215F, and lane 3 is the native ω -transaminase.

Detailed Description

Example 1: preparation and construction of omega-transaminase site-directed mutants

Omega-transaminase 1 site-directed mutant I215F from Bacillus pumilus W3:

in the invention, a three-dimensional simulation structure of Bacillus pumilus W3 omega-transaminase (omega-BPAT) is constructed by a Swiss-Model online server by taking a crystal structure (PDB ID:5E25) of the thermophilic archaea transaminase with the highest similarity as a template (FIG. 1). Through amino acid primary sequence alignment, the similarity between the thermophilic archaea transaminase and the omega-BPAT reaches 51.21 percent, and accords with the parameters of homology modeling, so that the omega-BPAT can be considered to have a three-dimensional structure similar to the thermophilic archaea transaminase. Based on the results predicted by software analysis, mutant I215F was constructed using PCR-mediated site-directed mutagenesis.

According to the preparation method of the site-directed mutant, primers for introducing site-directed mutation are respectively designed and synthesized according to the sequence (the amino acid sequence is shown as SEQ ID NO: 1) of Bacillus pumilus W3 omega-transaminase, site-directed mutation is carried out on the I215 position of the omega-transaminase, DNA coding sequences are determined, and the sequence is respectively determined to confirm whether the coding gene of the omega-transaminase mutant is correct or not; the mutant gene is connected to a proper expression vector (any one of pET series, pGEX series, pCold series or pUB) and is introduced into escherichia coli for expression, and the corresponding omega-transaminase site-directed mutant is obtained.

PCR amplification of site-directed mutant coding gene: using PCR technology, expression vector ota3/pCold II was used as template.

The mutation primers for introducing the I215F site-directed mutation are (SEQ ID NO:3 and SEQ ID NO:4, respectively):

I215F-F：5’-GGCGCATTAGAGGGTTTTACCCGCAACGCAA-3’

I215F-R：5’-AATATAACCTGGCGGAGTGTACAGTTTACC-3’

the PCR amplification program was set up as follows: first, pre-denaturation at 94 ℃ for 5 min; then 30 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 5 min; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel.

After the PCR product is purified, DPn I is added, water bath is carried out for 1h at 37 ℃, a template is degraded, then E.coli JM109 is transformed, positive clone is selected, L B liquid culture medium is cultured for 8-10h, a glycerol tube is kept, sequencing is carried out, a mutant with correct sequencing (the amino acid sequence is shown as SEQ ID NO:5, the nucleotide sequence is shown as SEQ ID NO: 2) is inoculated to L B culture medium from the glycerol tube, overnight culture is carried out, a plasmid is extracted, and the plasmid is transformed to express host escherichia coli B L21 (DE3) competent cells, so that the recombinant strain capable of expressing the mutant I215F is obtained.

Example 2: expression and purification of natural omega-aminotransferase and site-directed mutants thereof

Selecting a positive monoclonal transferred into an expression host escherichia coli B L21 (DE3), growing for 8-10h in L B liquid culture medium (containing 30 mu g/m L ampicillin), and inoculating according to 5 percentThe seed fermentation broth was inoculated into L B liquid medium (containing 30. mu.g/m L ampicillin) and E.coli was shake-cultured at 37 ℃ for 2 hours to OD₆₀₀The mutant I215F recombinant strain is added with IPTG with 0.05mM final concentration to induce extracellular expression, and after the strain is cultured and fermented continuously for 24h in a shaking table at 15 ℃, the fermentation liquor is centrifuged for 10min at 8000g and 4 ℃ to remove thalli, and the centrifuged fermentation supernatant is collected. Slowly adding 60% (NH) into the mutant fermentation supernatant under low-speed stirring by a magnetic stirrer₄)₂SO₄The method comprises the steps of standing at 4 ℃ for salting out overnight, centrifuging at 10000g for 20min, collecting precipitates, redissolving the precipitates by using a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 50 mmol/L of 5.3, dialyzing in a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 50 mmol/L of 4.3 for overnight after redissolving the precipitates, replacing 2-3 times of the dialysis buffer solution during the redissolving process by using a 0.22 mu M membrane, filtering to prepare a sample, purifying the recombinant protein by using an AKTA avant protein purifier, controlling the temperature in the whole purification process to be 4 ℃, purifying by using a cation exchange chromatography, wherein (1) a balance is that a strong cation exchange chromatographic column is balanced by using a 5-fold volume of 50 mmol/L of pH value of 5.3, a sample is previously treated at the flow rate of 1M L/min, and (3) elution comprises eluting unadsorbed substances, heteroproteins and target proteins, the flow rate of 1.0M L/min, wherein the eluent is 50/L mmol of NaCl, the pH value of 5.3, linear citrate-disodium hydrogen phosphate buffer solution, the eluent is eluted by using a linear electrophoresis test, and the peak of a native enzyme is detected by using a native enzyme, and the peak of a native enzyme, wherein the peak of a native enzyme, the electrophoresis test shows that the peak of a native enzyme, and the peak of.

Example 3: enzyme activity analysis method

The method for measuring the activity of ω -transaminase is described in Gao, S. (Gao, S., Su, y., Zhao, L., L i, g., Zheng, g.,2017. characteristics of a (R) -selective amine transferase from fusarium oxysporum. process. biochem.63, 130-136.).

An appropriate amount of the cell supernatant (or the diluted purified enzyme solution) was added to 500. mu. L sodium dihydrogen phosphate/disodium hydrogen phosphate buffer (100mM, pH7.0) containing 20mM (R) - α -phenylethynylamine (or (S) - α -phenylethynylamine), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (P L P), and the mixture was mixed, reacted at 45 ℃ for 15 minutes, and then the reaction was stopped by adding an equal amount of ethyl acetate to measure the absorbance at 254nm of the solution before and after the reaction.

The amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute under the above conditions is defined as one enzyme activity unit (U/ml). The process is illustrated by △ A₂₅₄Calculating the enzyme activity of omega-transaminase, U/ml (△ A/min) V/rvb, △ A/min-absorbance change, V-reaction system volume (ml), r-molar extinction coefficient (cm)²/umol); v-sample size (ml); b-cuvette optical path length (cm), the above amounts can be increased or decreased proportionally.

Through determination, the crude enzyme activity of the natural enzyme is 1.1760U/m L, and the enzyme activity of the recombinant omega-transaminase is 3.2988U/m L.

Example 4: determination of kinetic parameters of omega-transaminase mutants

The kinetic parameters of ω -transaminase were determined by Gao, S. (Gao, S., Su, y., Zhao, L., L i, g., Zheng, g.,2017.Characterization of a (R) -selective amine transferase from fusarium oxysporum. process. biochem.63, 130-136.).

In this example, the kinetic parameters of the natural enzyme (amino acid sequence shown in SEQ ID NO: 1) and the mutant I215F (amino acid sequence shown in SEQ ID NO: 5) purified in example 2 at 45 ℃ were determined by adding 500. mu. L of sodium dihydrogenphosphate/disodium hydrogenphosphate buffer (100mM, pH7.0) containing (R) - α -phenylethynamine at different concentrations (specific concentration gradient referred to above), 20mM sodium pyruvate, and 0.1mM pyridoxal 5' -phosphate (P L P) to a suitable cell supernatant (or a purified diluted enzyme solution), mixing the mixture, reacting the mixture at 45 ℃ for 15min, and adding the same amount of ethyl acetate to terminate the reaction, the results of the kinetic studies are shown in Table 1.

The results show that K of mutant I215F compared to the native enzyme (WT)_mThe value is reduced by 43.4%, K_mThe decrease in value indicates an increased affinity of the mutant for the substrate (R) - α -phenylethylamine, in addition, the catalytic constant K of I215F_catIs improved and is natural enzyme1.08 times. Catalytic constant K compared to the native enzyme_catAnd the increased affinity for the substrate, directly led to the catalytic efficiency K of the mutant I215F_cat/K_mThe improvement is 1.92 times.

As shown in fig. 1, I215F is far away from the catalytic center and the isomerization region of ω -transaminase, and mutation may cause the structure of the region other than the catalytic center and the isomerization region to become compact, so that the substrate is not easily detached from the catalytic center, thereby possibly having a positive effect on the kinetic parameters of the enzyme.

TABLE 1 kinetic parameters of the ω -transaminase mutants

The conversion rate is the conversion efficiency of (R) - α -phenylethynamine into acetophenone.

Example 5 application of the mutant in the production of (R) - α -phenylethylamine

In the embodiment, (R) - α -phenylethynylamine is taken as an amino donor, sodium pyruvate is taken as an amino acceptor, and the catalytic capability of the obtained recombinase and the mutant recombinase on the enzyme is detected, so that whether the enzyme can efficiently catalyze the (R) - α -phenylethynylamine is judged, and the application of the enzyme in the industrial synthesis of chiral amines is determined.

Chiral synthesis catalysis experiment, which is to take a proper amount of purified diluted enzyme solution, add 500 mu L sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution (100mM, pH7.0) containing 20mM (R) - α -phenylethynylamine, 20mM sodium pyruvate, 0.1mM pyridoxal 5' -phosphate (P L P), mix well, react for 15min at 45 ℃, then add equal amount of ethyl acetate to stop the reaction, centrifuge for 1min at 12,000 × g, take the upper organic phase, filter with 0.22 mu m filter membrane, and detect the product as acetophenone by high performance liquid phase (HP L C).

Detection conditions are as follows:

column Agilent C18 column (250X 4.6 mm, Agilent, USA), mobile phase acetonitrile/water (50/50, v/v), flow rate 0.6m L/min, detection wavelength 254 nm.

TABLE 2

And (3) comparison: and (R) -phenylethyylamine is used as a substrate, and the catalytic capability of the natural omega-transaminase on the (R) -phenylethyylamine is detected under the reaction conditions.

Test samples: the catalytic ability of the omega-transaminase I215F mutant was tested under the above reaction conditions using (R) -phenylethylamine as a substrate.

The data show that the catalytic activity of the recombinase on an experimental group (taking the omega-transaminase I215F mutant as a catalyst) is better than that of a control group (taking the natural omega-transaminase as a catalyst). The data show that the recombinant omega-transaminase I215F mutant has the function of efficiently and selectively synthesizing R-chiral amine and has larger application potential (see table 3).

TABLE 3

Enzyme activity (U/mg): the amount of enzyme required to catalyze 1. mu. mol of the relevant ketones in 1 minute is defined as one unit of enzyme activity (U/mg).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> an omega-transaminase mutant I215F with improved catalytic efficiency

<160>5

<170>PatentIn version 3.3

<210>1

<211>304

<212>PRT

<213> Artificial sequence

<400>1

Met Glu Asp Gln Lys Glu Gln Trp Ile Phe Leu Asn Asp Glu Leu Val

1 5 10 15

Lys Lys Glu Asp Ala Lys Ile Ser Val Tyr Asp HisGly Phe Leu Tyr

20 25 30

Gly Asp Gly Val Phe Glu Gly Ile Arg Val Tyr Asn Gly Asn Ile Phe

35 40 45

Arg Met Lys Glu His Leu Asp Arg Leu Tyr Asp Ser Ala Arg Ser Ile

50 55 60

Met Leu Asn Ile Pro Tyr Ser Leu Glu Glu Leu Thr Glu Lys Met Ile

65 70 75 80

His Thr Val Glu Arg Asn Gly Leu Lys Asp Ala Tyr Ile Arg Leu Val

85 90 95

Val Ser Arg Gly Ala Gly Asp Leu Gly Leu Asp Pro Asn Asn Cys Gly

100 105 110

Arg Ala Asn Thr Val Ile Ile Val Glu Pro Leu Ala Ile Phe Pro Lys

115 120 125

His Leu Tyr Glu Thr Gly Ile Asp Ile Val Thr Val Pro Thr Arg Arg

130 135 140

Asn Arg Pro Asp Val Leu Ser Pro Lys Val Lys Ser Leu Asn Tyr Leu

145 150 155 160

Asn Asn Ile Leu Val Arg Ile Glu Ala His Met Ala Gly Val Ser Glu

165 170 175

Ala Leu Met Leu Asn Asp Gln Gly Tyr Val Ala Glu Gly Ser AlaAsp

180 185 190

Asn Val Phe Ile Tyr Lys Lys Gly Lys Leu Tyr Thr Pro Pro Gly Tyr

195 200 205

Ile Gly Ala Leu Glu Gly Ile Thr Arg Asn Ala Ile Met Glu Ile Ala

210 215 220

Glu Asp Leu Gly Tyr Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp

225 230 235 240

Val Tyr Thr Ala Glu Glu Val Phe Leu Thr Gly Thr Ala Ala Glu Val

245 250 255

Ile Ala Val Val Lys Val Asp Gly Arg Met Ile Gly Glu Gly Lys Pro

260 265 270

Gly Phe His Thr Asn Lys Leu Leu Glu Gln Phe Arg Lys Arg Val Val

275 280 285

Glu Glu Gly Glu Lys Val Val Phe Thr Asp Glu Asn Ile His Ala Ser

290 295 300

<210>2

<211>915

<212>DNA

<213> Artificial sequence

<400>2

atggaggatc agaaggaaca gtggatcttt ctgaacgatg agctggtgaa gaaggaagac 60

gcaaaaatct ctgtatacga ccatgggttc ttatatggcg atggggtctt cgaaggcatt 120

cgtgtctaca acggcaatat cttccgtatg aaagaacatt tggaccgcct ttacgatagc 180

gcacgtagta ttatgttgaa tatcccgtat tcacttgaag agctgacaga aaagatgatt 240

cacacagttg agcgtaacgg tttgaaggat gcttacatcc gcttggtcgt gtctcgcgga 300

gcgggcgact tgggattaga cccgaacaat tgtggtcgcg ccaatactgt tatcattgtt 360

gaacctctgg caattttccc gaaacatttg tacgagaccg gcatcgacat tgtaacggtt 420

cccacccgcc gtaatcgtcc cgatgtcctt tcgccaaaag taaaatctct taattatctg 480

aacaacatcc ttgttcgcat tgaggcccac atggcaggtg taagtgaagc gttaatgctt 540

aatgatcaag gatatgtagc cgaagggtcg gctgataatg tttttatcta taaaaaaggt 600

aaactgtaca ctccgccagg ttatattggc gcattagagg gttttacccg caacgcaatt 660

atggagattg cggaagatct tgggtacgag gtcaaggaag agcctttcac gcgccatgac 720

gtctatacag cagaggaagt ttttcttacc ggtacggctg ctgaggtcat cgcagttgta 780

aaggttgacg gacgcatgat tggcgaagga aagccggggt tccacactaa caaattactt 840

gaacaattcc gcaaacgcgt agtggaggag ggagagaagg ttgtctttac ggatgaaaat 900

atccatgcgt cgtaa 915

<210>3

<211>31

<212>DNA

<213> Artificial sequence

<400>3

ggcgcattag agggttttac ccgcaacgca a 31

<210>4

<211>30

<212>DNA

<213> Artificial sequence

<400>4

aatataacct ggcggagtgt acagtttacc 30

<210>5

<211>304

<212>PRT

<213> Artificial sequence

<400>5

Met Glu Asp Gln Lys Glu Gln Trp Ile Phe Leu Asn Asp Glu Leu Val

1 5 10 15

Lys Lys Glu Asp Ala Lys Ile Ser Val Tyr Asp His Gly Phe Leu Tyr

20 25 30

Gly Asp Gly Val Phe Glu Gly Ile Arg Val Tyr Asn Gly Asn Ile Phe

35 40 45

Arg Met Lys Glu His Leu Asp Arg Leu Tyr Asp Ser Ala Arg Ser Ile

50 55 60

Met Leu Asn Ile Pro Tyr Ser Leu Glu Glu Leu Thr Glu Lys Met Ile

65 70 75 80

His Thr Val Glu Arg Asn Gly Leu Lys Asp Ala Tyr Ile Arg Leu Val

85 90 95

Val Ser Arg Gly Ala Gly Asp Leu Gly Leu Asp Pro Asn Asn Cys Gly

100 105 110

Arg Ala Asn Thr Val Ile Ile Val Glu Pro Leu Ala Ile Phe Pro Lys

115 120 125

His Leu Tyr Glu Thr Gly Ile Asp Ile Val Thr Val Pro Thr Arg Arg

130 135 140

Asn Arg Pro Asp Val Leu Ser Pro Lys Val Lys Ser Leu Asn Tyr Leu

145 150 155 160

Asn Asn Ile Leu Val Arg Ile Glu Ala His Met Ala Gly Val Ser Glu

165 170 175

Ala Leu Met Leu Asn Asp Gln Gly Tyr Val Ala Glu Gly Ser Ala Asp

180 185 190

Asn Val Phe Ile Tyr Lys Lys Gly Lys Leu Tyr Thr Pro Pro Gly Tyr

195 200 205

Ile Gly Ala Leu Glu Gly Phe Thr Arg Asn Ala Ile Met Glu Ile Ala

210 215 220

Glu Asp Leu Gly Tyr Glu Val Lys Glu Glu Pro Phe Thr Arg His Asp

225 230 235 240

Val Tyr Thr Ala Glu Glu Val Phe Leu Thr Gly Thr Ala Ala Glu Val

245 250 255

Ile Ala Val Val Lys Val Asp Gly Arg Met Ile Gly Glu Gly Lys Pro

260 265 270

Gly Phe His Thr Asn Lys Leu Leu Glu Gln Phe Arg Lys Arg Val Val

275 280 285

Glu Glu Gly Glu Lys Val Val Phe Thr Asp Glu Asn Ile His Ala Ser

290 295 300

Claims

1. An omega-transaminase mutant with improved catalytic efficiency is characterized in that the amino acid sequence of the omega-transaminase mutant is shown as SEQ ID NO. 5.

2. A recombinant plasmid vector comprising a nucleotide sequence encoding the mutant of claim 1.

3. The recombinant plasmid vector according to claim 2, wherein the recombinant plasmid vector is constructed on the basis of any one of the plasmid vectors of the pET series, the pGEX series, the pCold series, or the pUB series.

4. A gene encoding the mutant of claim 1.

5. A genetically engineered bacterium expressing the mutant of claim 1.

6. The mutant of claim 1, the gene of claim 4, the genetically engineered bacterium of claim 5, and the use of the mutant in preparing chiral amine or unnatural amino acid, wherein the use is for catalyzing (R) -phenylethylamine to generate acetophenone.

7. Use according to claim 6, for food, chemical or for the preparation of a medicament.