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A kind of ω-transaminase mutant based on co-evolution network and preparation method and application

A mutant and transaminase technology, applied in the field of molecular biology, can solve the problem that ω-transaminase needs to be further improved, and achieve the effect of increasing screening workload, increasing the probability of successful mutation, and improving experimental efficiency

Active Publication Date: 2020-08-25
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although the mutant obtained by the above-mentioned molecular modification method has improved the thermal stability of the wild-type ω-transaminase to a certain extent, the above-mentioned ω-transaminase still needs to be further improved.

Method used

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  • A kind of ω-transaminase mutant based on co-evolution network and preparation method and application
  • A kind of ω-transaminase mutant based on co-evolution network and preparation method and application
  • A kind of ω-transaminase mutant based on co-evolution network and preparation method and application

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Experimental program
Comparison scheme
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Embodiment 1

[0044] 1. Coevolutionary Network Prediction

[0045] 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.

[0046] Specific steps are as follows:

[0047] (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.

[0048] (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;

[0049] Upload the protein famil...

Embodiment 2

[0070] 1. Site-directed mutagenesis and expression and purification stages

[0071] Design primers for the 118th amino acid residue site, site-directed mutation into threonine (T), the mutant primers are as follows:

[0072] L118T-F: 5'-GGGATGCATTTGTTGAA ACC ATAGTCACCCGCGGTC-3';

[0073] L118T-R: 5'-GACCGCGGGTGACTAT GGT TTCAACAAATGCATCCC-3'.

[0074] The steps of subsequent PCR amplification, expression and purification stages are exactly the same as those in Example 1.

[0075] 2. Investigation of the thermodynamic stability of mutants

[0076] 1) Half inactivation temperature (T 50 10 ) refers to the temperature at which half of the enzyme activity is lost after the enzyme is incubated at a specific temperature for 10 minutes, which is an important parameter to characterize the thermal stability of the enzyme.

[0077] The wild-type ω-transaminase and the mutant ω-transaminase were respectively placed in a water bath at 25, 30, 35, 40, 45, 50, and 55°C for 10 minutes...

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Abstract

The invention discloses an omega-transaminase mutant based on co-evolution network and a preparation method and application thereof. The omega-transaminase mutant has an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 3. The omega-transaminase mutant has half inactivation temperature of 42.2 DEG C and 43.8 DEG C which is 3.7 DEG C and 5.3 DEG C higher than that of the wild enzyme, has a half-life period of 20.6 min and 26.0 min at 40 DEG C, which is 13.7-19.1 min longer than that of the wild enzyme. The protein co-evolution network is used herein to guide the research on omega-transaminase; biological informatics and computer software are combined to predict an important locus that affects the structure and functionality of omega-transaminase and construct a mutant library so as toobtain a mutant with further improved enzymic activity and thermal stability; mutation success rate is effectively increased; screening workload is reduced; experimental efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to an omega-transaminase mutant based on a co-evolution network, a preparation method and an application. Background technique [0002] Transaminase is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that exists widely in nature and can catalyze the transfer reaction of amino groups from amino donors to amino acceptors. play an important role. As a biocatalyst, transaminase can be used to catalyze the preparation of chiral amine compounds, as an important intermediate in the synthesis of diabetes drugs, cardiovascular drugs, neurological drugs, antihypertensive drugs and anti-infective drugs. [0003] According to the multiple sequence alignment in the PFAM database, transaminases can be divided into 5 categories: aspartate aminotransferase, aromatic transaminase, ω-transaminase, branched-chain transaminase and D-transaminase. Since the substrate binding pocket of ω-tran...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/10
CPCC12N9/1096C12N15/1031C12Y206/01C12Q2531/113
Inventor 黄俊梅乐和朱婉丽吕常江胡升赵伟睿王宏鹏
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY