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Omega-transaminase mutant based on ancestor sequence reconstruction

A transaminase, mutant technology, applied in the field of molecular biology, to achieve the effect of improving thermal stability

Active Publication Date: 2022-03-04
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] At present, there is no relevant research report on improving the thermal stability of Aspergillus terreus ω-transaminase by using the ancestral sequence reconstruction technology to obtain the ancestral enzyme of the existing enzyme

Method used

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  • Omega-transaminase mutant based on ancestor sequence reconstruction
  • Omega-transaminase mutant based on ancestor sequence reconstruction
  • Omega-transaminase mutant based on ancestor sequence reconstruction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The amino acid sequence of the wild-type ω-transaminase from Aspergillus terreus is shown in SEQ ID No.2, and the gene sequence is shown in SEQ ID No.1.

[0032]The protein sequence of Aspergillus terreus ω-transaminase was uploaded to Fire ProtASR (https: / / loschmidt.chemi.muni.cz / fireprotasr / , a server for automatic ancestral sequence reconstruction), and the Aspergillus terreus ω -The phylogenetic tree of transaminase, and then select each node on the evolutionary tree that finally evolved into the branch of Aspergillus terreus ω-transaminase, and download the gene sequences corresponding to these nodes from this website, and then obtain the corresponding mutant gene by whole gene synthesis sequence. After enzyme expression and purification and thermal stability testing, two mutants with significantly improved thermal stability were finally obtained, named Ancata-101 and Ancata-124 respectively. The mutation sites and sequences are as follows:

[0033] Ancata-101: D5...

Embodiment 2

[0036] (1) Materials and reagents

[0037] (R)-ω-TA and the whole mutant gene were synthesized by Anhui General Biology Co., Ltd., the vector was pET-28a(+), and the expression host strain was E.coli BL21(DE3); isopropyl-β-D-thio Galactoside (IPTG), Kanamycinsulfate (Kanamycinsulfate), pyridoxal-5'-phosphate (PLP), and improved Bradford protein concentration assay kits were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. ; Protein Marker and Ni-NTA chromatography media were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit was purchased from Kangwei Century Biotechnology Co., Ltd. Ltd.; dimethyl sulfoxide (DMSO), pyruvate, and (R)-α-methylbenzylamine were purchased from Aladdin Biochemical Technology Co., Ltd.

[0038] (2) Expression and purification of enzymes

[0039] Take 10 μL of the wild-type recombinant plasmid bacterial liquid and the mutant bacterial liquid and...

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Abstract

The invention discloses an omega-transaminase mutant based on ancestor sequence reconstruction, and relates to the technical field of molecular biology. The mutant is obtained by mutation of omega-transaminase from Aspergillus terreus, and the amino acid sequence of the omega-transaminase mutant is as shown in SEQ ID NO. 4 or SEQ ID NO. 6. The invention further discloses a preparation method of the mutant. Compared with a wild type enzyme, the half-life period of the omega-transaminase mutant is more than 24 hours, the half-life period of the wild type enzyme is only 6.90 minutes, the half-inactivation temperatures of the mutant are 49.00 DEG C and 49.03 DEG C respectively and are about 11 DEG C higher than that of the wild type enzyme (37.89 DEG C), and the thermal stability is remarkably improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant based on ancestral sequence reconstruction. Background technique [0002] Chiral amine refers to a class of compounds containing amino groups in the chiral center of small molecular compounds, and is an important class of intermediates in drug synthesis. In recent years, with the continuous expansion of the chiral drug market, chiral amines and their derivatives account for more than 70% of chiral drugs, such as neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines; The main component of diabetes medicine sitagliptin is R-amine. The market demand for chiral amine drugs is huge, making the efficient preparation of chiral amine drugs extremely important. [0003] Transaminase is the key enzyme for the biological preparation of chiral amines, which can reversibly catalyze the transamination reacti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P7/26
CPCC12N9/1096C12Y206/01C12P7/26
Inventor 黄俊蔡婷婷邱帅吕常江樊芳芳李业王丰李元源
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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