A kind of ω-transaminase double mutant and its application

A double mutation, transaminase technology, applied in the field of molecular biology

Active Publication Date: 2021-02-19
山东聚鸿生物科技研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the improvement of the stability of the enzyme activity by the mutation of the single mutation site is limited. In order to further improve the thermal stability of the transaminase, it is necessary to introduce mutations at other sites.

Method used

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  • A kind of ω-transaminase double mutant and its application
  • A kind of ω-transaminase double mutant and its application
  • A kind of ω-transaminase double mutant and its application

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Experimental program
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Embodiment 1

[0034] 1. Co-evolution network prediction

[0035] Protein coevolution is of great significance for gaining insight into the action process and protein structure of functional proteins. Through the measurement of protein co-evolution, residue sites that play an important role in protein structure and function can be found.

[0036] Specific steps are as follows:

[0037] (1) The PDB file (PDB ID: 4CE5) of Aspergillus terreus (Aspergillus terreus) ω-transaminase was compared in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) through BLASTP multiple sequence alignment to obtain Related protein family number pfam:01063.

[0038] (2) Based on mutual information (Mutual Information, MI), through different biological characteristics, the protein co-evolution website MISTIC (Mutual Information Server To Infer Coevolution) is used to infer protein coevolution through mutual information, and an amino acid residue co-evolution network is generated;

[0039]Upload the protein family...

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PUM

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Abstract

The invention discloses an omega-aminopherase double mutant and an application thereof. The amino acid sequence of the omega-aminopherase double mutant is as shown in SEQID NO.1. The omega-aminopherase double mutant disclosed by the invention is obtained in a manner than the 115th phenylalanine of wild type aspergillus terreus (Aspergillus terreus) omega-aminopherase is mutated into leucine and the 118th leucine is mutated into threonine. The semi-inactivated temperature of the omega-aminopherase double mutant is 8.8 DEG C higher than that of wild type omega-aminopherase, the thermolysis collapse temperature of the omega-aminopherase double mutant is increased by 7.7 DEG C than that of the wild type omega-aminopherase, and the half-life period of the omega-aminopherase double mutant at 40DEG C is prolonged by 59.0min than that of the wild type omega-aminopherase and is 9.55 times of that of the wild type omega-aminopherase.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an ω-transaminase double mutant and its application. Background technique [0002] Amine transaminases (ATAs) have become the most important biocatalysts for the preparation of optically pure chiral amines due to their high stereoselectivity and mild reaction conditions. Generally speaking, increasing the temperature can increase the solubility of raw materials and products, reduce their viscosity, and speed up the reaction speed. However, the temperature tolerance of enzymes in the natural state is poor, and high temperatures can easily inactivate and denature enzymes. How to improve the thermal stability of enzymes Engineering challenging studies. [0003] The site-directed mutagenesis technology invented by Michael Smith is to insert, replace and delete specific nucleotides in the known gene sequence, thereby changing the primary structure of the protein. Zhang Xia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12P7/26
CPCC12N9/1096C12P7/26C12Y206/01
Inventor 黄俊梅乐和吕常江朱婉丽王宏鹏胡升赵伟睿
Owner 山东聚鸿生物科技研究院有限公司
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