Omega-transaminase mutant and application thereof

A mutant and transaminase technology, applied in the field of molecular biology, can solve the problems of half-inactivation temperature and half-life to be further improved, and achieve the effects of improving experimental efficiency and feasibility, improving thermal stability, and increasing the probability of positive mutation

Active Publication Date: 2018-11-30
杭州快格科技有限公司
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Problems solved by technology

[0011] Although the above-mentioned mutants can improve the half-inactivation temperature and prolong the half-life compared with the wild-type ω-transaminase; but, if th...

Method used

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

Examples

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Embodiment 1

[0049] 1. Sequence Consistency Analysis

[0050] According to the ω-transaminase gene sequence of Aspergillus terreus in the NCBI database (Genbank: XM_001209325) and the codon usage frequency distribution table of Escherichiacoli in the codon usage data (http: / / www.kazusa.or.jp / codon / ), analyze ω - Codon usage for transaminases. Codon-optimized ω-transaminase gene (ω-opt-TA gene), its nucleotide sequence is shown in SEQ ID NO.3, its encoded protein has a total of 325 amino acids, and its amino acid sequence is shown in SEQ ID NO.4 .

[0051] Through BLAST comparison, set the maximum value of E-value to 10 -3 , the sequence redundancy does not exceed 0.9, and a total of 91 homologous amino acid sequences were screened. Mutation sites were screened based on sequence consistency. The principle of screening was that the conservation threshold of mutations was 0.6, that is, the amino acid type of this site accounted for more than 60% in the multiple sequence alignment, and the ...

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Abstract

The invention discloses a Omega-transaminase mutant and an application thereof. The amino acid sequence of the Omega-transaminase mutant is shown as SEQ ID NO.2. The Omega-transaminase mutant disclosed by the invention is acquired by respectively mutating isoleucine on locus 77, methionine on locus 150, histidine on locus 210 and methionine on locus 280 of aspergillus terreus Omega-transaminase into leucine, cysteine, asparaginate and cysteine. Semi-inactivation temperature (T5010) of the Omega-transaminase mutant is at 50.3 plus or minus 0.5 DEG C and is 11.8 DEG C higher than that of wild type; the half-life period (t1/2) at 40 DEG C is 114.6 plus or minus 2.8 min and is 16.6 times of that of wild type; heat stability is obviously promoted.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant and its application. Background technique [0002] Chiral amine compounds are important pharmaceutical and fine chemical intermediates. At present, more than 70% of drugs, such as neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines, are synthesized using chiral amines as intermediates. [0003] The ω-transaminase from Aspergillus terreus uses ketones as raw materials, through stereoselective transamination, can efficiently produce chiral amines, and catalyze the transfer of amino groups from amino donors to prochiral acceptors Ketones, to obtain chiral amines and by-product ketones, the reaction process requires the participation of pyridoxal phosphate (pyridoxal phosphate, PLP), the catalytic process is as follows: [0004] [0005] Studies have shown that the half-life of the wild-type e...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/26C12R1/19
CPCC12N9/1096C12N15/70C12P7/26C12Y206/01
Inventor 黄俊梅乐和谢东芳吕常江朱婉丽胡升赵伟睿
Owner 杭州快格科技有限公司
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