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Carboxylic acid reductase mutant with improved catalytic activity and coding gene, genetically engineered bacterium and application thereof

A technology of improving catalytic activity and encoding genes, which is applied in the field of protein engineering, can solve the problems of low catalytic activity and low yield rate, and achieve the effect of high-efficiency biocatalysis and activity improvement

Active Publication Date: 2021-11-05
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a carboxylic acid reductase mutant with improved catalytic activity and its coding gene, genetically engineered bacteria and application, thereby solving the catalytic activity of carboxylic acid reductase to a series of organic acids such as vanillic acid in the prior art Low, leading to the problem of low space-time yield in the catalytic process

Method used

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  • Carboxylic acid reductase mutant with improved catalytic activity and coding gene, genetically engineered bacterium and application thereof
  • Carboxylic acid reductase mutant with improved catalytic activity and coding gene, genetically engineered bacterium and application thereof
  • Carboxylic acid reductase mutant with improved catalytic activity and coding gene, genetically engineered bacterium and application thereof

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Embodiment 1

[0026] Example 1 Substrate docking and molecular dynamics simulation

[0027] In order to obtain the spatial structure of the adenylation domain of carboxylic acid reductase MsCAR, homology modeling was performed on the domain. Modeling was performed using the SWISS-MODEL online server (http: / / www.swissmodel.expasy.org / ), and the 3D model of the adenylation state of the domain was based on the carboxylic acid reductase (PDB number: 5MSC) from Nocardia iowensis The structure was obtained as a template, and the 3D model of the thioesterification state was obtained using the structure of carboxylic acid reductase from Mycobacterium marinum (PDB code: 5MSS) as a template. The structure-optimized vanillyl-AMP complex was then docked into the structure of the adenylation state of the MsCAR adenylation domain. The docking results of AMP in the carboxylic acid reductase crystal structure 5MSC were screened, and the best docking posture was selected in combination with the docking s...

Embodiment 2

[0030] Example 2 Virtual mutation of the hinge region of carboxylic acid reductase MsCAR and its screening

[0031] Based on the structure of the adenylation state of the adenylation domain of carboxylic acid reductase MsCAR and the structure of the vanillyl-AMP complex, the point mutation of the protein was carried out in the Build and EditProtein program under the Macromolecules module in the Discovery Studio 2020 software, namely Select the site to be mutated in the structure, select the target amino acid in the Choose AminoAcid operation column to complete the amino acid mutation, and then call the Minimize and Refine Protein program under this module to optimize the structure of the mutated protein. After the optimization is completed That is to say, a more reasonable protein-vanillyl-AMP complex binding conformation has been obtained. Then call the interatomic distance measurement function in the Discovery Studio 2020 software to calculate the distance between the side...

Embodiment 3

[0033] Construction and screening of embodiment 3MsCAR mutants

[0034] The carboxylic acid reductase MsCAR producing strain was cultured in a large test tube for 10-12 hours, and its plasmid was extracted as a template for subsequent PCR. The KOD-Plus point mutation kit of Japan Toyobo Co., Ltd. was used to construct the mutation library. The specific operation is as follows: 1. According to the product manual, use the target sequence as a template to design primers; 2. Use high-fidelity KOD–Plus-enzyme to introduce point mutations by reverse PCR; 3. Use Dpn I to template plasmid DNA. Digest; 4. Use T4Polynucleotide Kinase and Ligation high in the kit to self-circularize the PCR product; 5. Transform and introduce the resulting cyclized product into E. coli BL21 (DE3) competent cells; 6. Pick a single colony And inoculated in 5mL LB medium, cultivated overnight at 37°C and then sequenced the strains; 7. After ensuring the sequence was correct, each mutant was induced to ex...

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Abstract

The invention discloses a carboxylic acid reductase mutant with improved catalytic activity and a coding gene, a genetically engineered bacterium and application thereof. Wild carboxylic acid reductase as shown in SEQ ID NO.1 is taken as a basis, and site-specific mutagenesis is carried out as follows: mutants R505E, R505I, R505M and R505Q formed by R mutation at the 505th site; a mutant N506K formed by N mutation at the 506th site; and mutants R505I / N506K, R505F / N506G, R505F / N506A, R505F / N506C, R505F / N506V, R505I / N506R, R505K / N506M, R505T / N506S and R505Y / N506E formed by simultaneous R mutation at the 505th site and N mutation at the 506th site. The mutants obviously improve the activity of catalyzing vanillic acid to vanillic aldehyde, and have wide application prospects.

Description

technical field [0001] The invention relates to the technical field of protein engineering, in particular to a carboxylic acid reductase mutant with improved catalytic activity and its application. Background technique [0002] Aldehydes are key intermediates in medicinal chemistry. The high reactivity of aldehydes is a desirable property for synthetic chemists, but also poses challenges for their synthesis. The classic method of synthesizing aldehydes is mostly achieved by oxidation at specific reaction centers, for example: NaClO / TEMPO / Co(OAc) can be used 2 System or IBX oxidizes the benzyl position; the terminal double bond can be replaced by O under Wacker conditions 2 Efficient oxidation; ozonation can convert alkenes to aldehydes in a highly atom-economical manner, but requires post-reductive post-treatment of the resulting ozonides; hydration of terminal triple bonds by Ru catalysts can also generate product aldehydes in good yields; The chemical conversion of carb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N1/21C12P7/24C12R1/19
CPCC12N9/0008C12P7/24Y02A50/30
Inventor 王华磊魏东芝王留柱
Owner EAST CHINA UNIV OF SCI & TECH
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