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Omega-transaminase mutant and its coding gene and preparation method

A mutant, transaminase technology, applied in the field of molecular biology, can solve the problem of further improvement of heat resistance, and achieve the effects of improving experimental efficiency and feasibility, increasing the probability of successful mutation, and excellent thermal stability.

Active Publication Date: 2018-09-11
ZHEJIANG ORIENT GENE BIOTECH
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Problems solved by technology

Experiments show that the half-life of the wild-type enzyme at 40°C is only 6.9 minutes, and its heat resistance needs to be further improved

Method used

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  • Omega-transaminase mutant and its coding gene and preparation method
  • Omega-transaminase mutant and its coding gene and preparation method
  • Omega-transaminase mutant and its coding gene and preparation method

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Embodiment Construction

[0050] The present invention will be further described below in conjunction with the examples. The specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in the claims. For the experimental methods not indicated in the implementation of the present invention, such as competent cell preparation, transformation and LB medium preparation, etc., refer to the third edition of "Molecular Cloning Experiment Guide" (J. Sambrook, D.W. Russell, Huang Peitang translation, The method in Science Press, 2002).

[0051] 1. Mutant construction

[0052] According to the ω-aminotransferase gene sequence of Aspergillus terreus NIH2624 in the NCBI database (Genbank: XM_001209325) and the Escherichia coli codon usage frequency distribution table in the codon usage database (http: / / www.kazusa.or.jp / codon / ), the analysis Codon usage of omega-transaminases...

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Abstract

The invention discloses an omega-transaminase mutant and an encoding gene and preparation method thereof. The amino acid sequence of the omega-transaminase mutant is shown in SEQ ID NO.2 or SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8 or SEQ ID NO.10. The nucleotide sequence of the omega-transaminase mutant is shown in SEQ ID NO.1 or SEQ ID NO.3 or SEQ ID NO.5 or SEQ ID NO.7 or SEQ ID NO.9. The invention further discloses an expression box, recombinant plasmid and converter containing the gene. Mutation site prediction is conducted on a wild omega-transaminase mutant through the combination of a temperature factor (B-factor) and the energy optimizing strategy, then site-specific mutagenesis is conducted, and the omega-transaminase mutant is prepared. The thermal stability of the selected mutant is higher than that of wild transaminase.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant, its coding gene and its preparation method. Background technique [0002] The method of stable evolution of enzymes has been one of the most important research fields in protein engineering. In recent years, computer-aided protein stabilization design has made breakthrough progress. Compared with the traditional random mutation evolution method, the computational aided design method based on energy calculation can improve the effectiveness of amino acid mutation, reduce the number of mutations, reduce the workload of screening, and greatly improve the success rate of design. As far as the thermostability modification of enzyme molecules is concerned, a variety of design methods have been applied to identify unstable regions of enzymes, and have been successfully applied in the thermostability modification of some enzymes. The temperature ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21
CPCC12N9/1096C12Y206/01
Inventor 黄俊谢东芳楼坚蒋成君吴元锋龚金炎
Owner ZHEJIANG ORIENT GENE BIOTECH
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