A mutant protein of enoate reductase and its application

A technology of mutants and reductases, which is applied in the field of directed evolution transformation of enzymes and biocatalysis applications, and can solve problems such as low catalytic efficiency

Active Publication Date: 2021-08-10
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a mutant protein of enoate reductase, which evolves through semi-rational design, aiming at the low catalytic efficiency of the existing enoate reductase ER-BC to 2-ene adipate Transformation, with higher enzyme activity; the mutant protein of the enoate reductase is applied to catalyze the reduction of enoic acid substances with higher catalytic efficiency

Method used

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  • A mutant protein of enoate reductase and its application
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  • A mutant protein of enoate reductase and its application

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Experimental program
Comparison scheme
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Embodiment approach

[0060] The existing enoate reductase ER-BC has a low catalytic efficiency to 2-ene adipate. In order to solve this problem, the inventors have conducted a lot of research on the enoate reductase ER-BC, and through semi-rational design evolution, A mutant protein of enoate reductase ER-BC with higher activity was obtained.

[0061] Furthermore, based on the enoate reductase gene (GenBank accession number CP003056.1) of Bacillus coagulans 36D1, the present inventors used error-prone PCR and semi-rational design methods, through random mutation and site-directed mutation, to Evolution has resulted in a mutant protein of the enoate reductase ER-BC.

[0062] Specifically, the inventors obtained a mutant protein of enoate reductase ER-BC with higher activity through the following steps:

[0063] (1) Based on the enoate reductase gene (GenBank accession number CP003056.1) of Bacillus coagulans 36D1, a random mutation library was constructed by error-prone PCR, and mutants with impro...

Embodiment 1

[0113] Example 1: Construction of error-prone PCR mutation library

[0114] Using the error-prone PCR kit (GeneMorph II Random Mutagenesis Kit (Agilent Technologies)), construct an Error prone with a base mutation rate of 1-2‰ (2-4 bases / gene) and an amino acid mutation rate of 1-2 amino acids / gene PCR mutation library.

[0115] The error-prone PCR reaction system is shown in Table 1, with a total volume of 50 μL.

[0116] Table 1 Error-prone PCR reaction system

[0117]

[0118] Wherein, the F and R primer sequences are:

[0119] Upstream primer F: 5′-CGCGGATCCATGAAGTACAAGAAGCTGTTCG-3′

[0120] Downstream primer R: 5′-CCCAAGCTTTTACAGGTTTGCAGCGACC-3′

[0121] The amplification program of error-prone PCR is shown in Table 2:

[0122] Table 2 Amplification program of error-prone PCR

[0123]

[0124]

[0125] After the error-prone PCR, the PCR products were separated and checked by agarose gel electrophoresis, and the gene DNA fragments required for the experiment...

Embodiment 2

[0126] Example 2: Screening of High Enzyme Activity Mutants in Error-prone PCR Mutation Library

[0127] It is known from the literature that NADH is required for ER-BC catalysis (Ress T, HummelW, Hanlon S.P, etal. ChemCatChem 2015, 7, 1302-1311), so it can be determined by UV / Vis-based method (UV / Vis absorption spectroscopy) The reduced amount of NADH is used for high-throughput screening of high-enzyme activity mutants.

[0128] 96-well plate primary screening:

[0129] (1) Prepare a sterilized 96 deep-well plate and HB-PET self-induction medium in advance, and fill the 96-deep-well plate with HB-PET self-induction medium with 0.1% Kana antibiotics in the ultra-clean bench, 700 μL per well;

[0130] (2) Label the single clones on the plate, then pick them into 96 deep-well plates one by one, and culture them at 30°C / 180rpm for 24h. Each deep-well plate has 6 unmutated single clones as a control;

[0131] (3) After the cultivation, put the 96 deep-well plate containing the...

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Abstract

The invention relates to a mutant protein of enoate reductase and its application. The mutant protein of the enoate reductase is obtained based on the enoate reductase gene of Bacillus coagulans 36D1, using error-prone PCR and semi-rational design methods, through random mutation and site-directed mutation, and through directed evolution; when The sequence comprises the amino acid mutants p.Ser109Asn, p.Gly111Asp, p.Leu181Pro, p.Ile187Asn, p. When one or several of p.Phe207Tyr, p.Phe229Ile, p.Gly531Ser, the enzymatic activity of the enoate reductase mutant is significantly improved compared with the starting strain.

Description

technical field [0001] The invention belongs to the technical field of directed evolution transformation of enzymes and biocatalytic application, and relates to a mutant protein of enoate reductase and its application. Background technique [0002] Adipic acid is often used as an intermediate in the synthesis of nylon 6.6 and is one of the most important dicarboxylic acids. It is estimated that by 2022, the global adipic acid market will reach 8 billion pounds, so the commercial value of adipic acid cannot be underestimated. At present, the production of adipic acid is generally carried out through petrochemical routes, such as the chemical synthesis of KA oil (cyclohexanol / cyclohexanone mixture) catalyzed by nitric acid, and a large amount of N will be produced during the synthesis process. 2 O, which causes more greenhouse climate impact than CO 2 298 times higher. N from adipic acid production 2 O emissions as a share of total industrial N 2 O emissions are about 80%...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12P7/44
CPCC12N9/001C12P7/44C12Y103/01031
Inventor 谭天伟李小芳陈必强张世鼎
Owner BEIJING UNIV OF CHEM TECH
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