Omega-transaminase mutant and application thereof

A transaminase and mutant technology, which is applied to ω-transaminase mutants and their application fields to achieve the effects of increasing the probability of positive mutation, improving experimental efficiency and feasibility

Active Publication Date: 2021-02-12
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is no related research on improving the thermal stability of Aspergillus terreus ω-transaminase by calculating the difference between the charge-charge interaction energy on the surface of the enzyme protein and the root mean square deviation (RMSD) by yasara simulation to report

Method used

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

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Experimental program
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Embodiment 1

[0030] (1) Materials and reagents

[0031] The whole gene of (R)-ω-TA was synthesized by Anhui General Biological Company, and the recombinant plasmid pET-28a-ω-TA was preserved by our laboratory. PrimeSTAR Max DNA polymerase was purchased from TaKaRa Company; Dpn I was purchased from Thermo Scientific Company; SanPrep column plasmid DNA mini-extraction kit, isopropyl-b-D-thiogalactoside (IPTG), kanamycin sulfate (Kanamycin sulfate), pyrodoxal phosphate (pyrodoxal-5'-phosphate, PLP), and improved Bradford protein concentration assay kit were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; DNA Marker, Protein Marker, E.coli BL21 (DE3), E.coli DH5α, and Ni-NTA chromatography media were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dansyl chloride (DNS-C1) was purchased from Sigma (USA); γ-aminobutyric acid standard ( γ-aminobutyric acid) was purchased from Fluka (Switzerland); sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel prepa...

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PUM

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Abstract

The invention discloses an omega transaminase mutant which is obtained by mutation of omega transaminase from Aspergillus terreus, the amino acid sequence of wild type omega transaminase is shown as SEQ ID No.2, and the mutation site of the omega transaminase mutant is at least one of E133Q, D224K, D231A, E253A or D268K. The omega transaminase mutant screened by the invention is obviously superiorto wild enzymes in thermodynamic stability and enzyme activity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an omega-transaminase mutant and its application. Background technique [0002] Chiral amines are an important part of chiral drugs and an extremely important class of fine chemical and pharmaceutical intermediates. With the continuous expansion of the chiral drug market, the demand for chiral amines is growing rapidly. Carrying out more in-depth research on chiral amines has greater commercial and economic benefits and industrial 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 is a type of enzyme that catalyzes the transfer of a certain group from a donor compound to an acceptor compound. It can reversibly transfer the amino group from the amino aci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/26C12R1/19
CPCC12N9/1096C12N15/70C12P7/26C12Y206/01Y02P20/584
Inventor 黄俊曹佳仁梅乐和陈海滨吕常江王宏鹏胡升赵伟睿李业樊芳芳于林凯周一峰
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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