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Omega-transaminase mutant, gene and application

A mutant, transaminase technology, applied in the field of molecular biology

Active Publication Date: 2020-10-27
李元源 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no relevant research report on improving the thermal stability of pergillus terreus ω-transaminase by computationally predicting the charge-charge interaction energy on the surface of the enzyme protein

Method used

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  • Omega-transaminase mutant, gene and application
  • Omega-transaminase mutant, gene and application
  • Omega-transaminase mutant, gene and application

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Embodiment 1

[0035] (1) Materials and reagents

[0036] 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-Cl) 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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Abstract

The invention discloses an omega-transaminase mutant, a gene and an application. The amino acid sequence of the wild type omega-transaminase is shown as SEQ ID No.2, and the omega-transaminase mutantis one of the follows: (1) an L118T / F115L dual-mutation mutant, (2) an L118T / F115L / E133A three-mutation mutant, (3) an L118T / F115L / E133K three-mutation mutant, and (4) L118T / F115L / E253A three-mutationmutant. Based on a genetic algorithm mature in technique, and a TK-SA model algorithm, improvement is performed, and then an ETSS algorithm is obtained; through combination with omega-transaminase surface charge-charge interaction, an amino acid residue site needing mutation is determined, and experimental verification is performed through a fixed-point mutation technique. The method can effectively improve orthomutation probability and improve experiment efficiency and feasibility, and through screening, a mutation enzyme of which the thermodynamics stability and the enzymatic activity are obviously better than those of a wild enzyme can be obtained.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant, gene and 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 fr...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P7/26C12R1/19
CPCC12N9/1096C12P7/26C12Y206/01
Inventor 花雨娇黄俊曹佳仁梅乐和吕常江王宏鹏于林凯周一峰葛青樊芳芳李业叶青文陈思艺张仪荻
Owner 李元源
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