Omega-transaminase mutant and preparation method and application thereof

A mutant, transaminase technology, applied in the field of molecular biology, can solve the problems of further improvement of thermal stability, unfavorable industrial production, etc., to achieve the effects of improving experimental efficiency and feasibility, and increasing the probability of positive mutation

Active Publication Date: 2017-08-18
上海邦林生物科技有限公司
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Problems solved by technology

[0005] Experiments have shown that the half-life of wild type ω-transaminase at 40°C is only 6.9 minutes, which is not conducive to the application in industrial production, and its therm

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  • Omega-transaminase mutant and preparation method and application thereof
  • Omega-transaminase mutant and preparation method and application thereof
  • Omega-transaminase mutant and preparation method and application thereof

Examples

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[0041] The present invention will be further described below in conjunction with embodiment. 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 protection scope of the claims.

[0042] For experimental methods not indicated in the examples, 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, translated by Huang Peitang, Science Publishing House, 2002) method.

[0043] DpnI enzyme was purchased from Thermo Scientific Company, and PrimeStar Max DNA polymerase was purchased from TaKaRa Company.

[0044] The oligonucleotide sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0045] Plasmid extraction, gel recovery kits, PCR cleaning kits, and SDS-PAGE gel preparation kits were purchased from Kangwei Century Compa...

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Abstract

The invention discloses an omega-transaminase mutant and a preparation method and application thereof and belongs to the technical field of molecular biology. The amino acid sequence of the omega-transaminase mutant is as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12. The invention further provides the gene of the omega-transaminase mutant, an expression unit containing the gene, recombinant plasmid and a transformant. The preparation method includes: using an NCBI database and BLAST software to perform comparison screening to obtain the homologous amino acid sequence of omega-transaminase, using a Weblogo program to obtain a sequence consistency result, using the homologous amino acid sequence, the sequence consistency result and the sequence of the omega-transaminase to determine amino acid residue sites which need to be mutated, and using site-directed mutagenesis technology to perform experimental verification. The method has the advantages direct mutation probability can be increased effectively, experiment efficiency and feasibility are increased, and mutant enzymes whose thermodynamic stability is evidently better than that of wild enzymes can be screened out.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant and its preparation method and application. Background technique [0002] Chiral amines are a class of valuable pharmaceutical and fine chemical intermediates. In-depth research on the asymmetric synthesis of chiral amines has great economic benefits and application value. At present, more than 70% of drugs are chiral amines and their derivatives. For example, the synthesis of neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines uses chiral amines as intermediates. [0003] Transaminase can not only split racemic amines through kinetics, but also generate chiral amines through asymmetric synthesis of ketones. Compared with traditional chemical catalytic methods, transaminases are more attractive and competitive, and have become industrially used to produce amino acids and chiral amines. One of ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12P7/26
Inventor 黄俊谢东芳方卉梅乐和王宏鹏胡升吕常江赵伟睿
Owner 上海邦林生物科技有限公司
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