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A kind of nitrilase mutant with improved reaction specificity and application thereof

A nitrilase and mutant technology, applied in the field of enzyme engineering, can solve the problems that mutants cannot be applied to industrial biocatalytic processes, nitrile hydration activity enhancement or incomplete elimination, etc.

Active Publication Date: 2021-08-24
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above studies provide a useful reference for the regulation of nitrilase reaction specificity, but the mutants cannot be used in industrial biocatalytic processes due to the incomplete enhancement or elimination of nitrile hydration activity

Method used

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  • A kind of nitrilase mutant with improved reaction specificity and application thereof
  • A kind of nitrilase mutant with improved reaction specificity and application thereof
  • A kind of nitrilase mutant with improved reaction specificity and application thereof

Examples

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Effect test

Embodiment 1

[0033] 1. Selection of nitrilase mutation sites

[0034]When rice nitrilase (OsNIT, GenBank accession number: AB027054, amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) catalyzes the reaction of benzyl nitrile, the ratio of amide and carboxylic acid in the product is close to 1:1, Such as figure 1 shown. Through bioinformatics analysis, the present invention determines that its catalytic triplet is 196Cys-71Glu-162Lys, and further screens out 18 amino acid residues near the catalytic pocket that may affect the specificity of the reaction: A72, A167, F175, E198, Y77, S229, T166, E169, I195, R224, S230, W197, H159, N199, K200, T220, A221 and V226.

[0035] By performing site-directed saturation mutations on the above amino acid residues, six mutation sites Y77, I195, W197, N199, K200, T220, R224 and V226 that have a greater impact on the specificity of the catalytic reaction were obtained, including Y77, K200 , R224 and V226 site mutations of the nitrilase mu...

Embodiment 2

[0057] 1. Construction and expression of nitrilase multiple mutants

[0058] Using the mutant plasmid with excellent reaction specificity or high enzyme activity obtained in Example 1 as a template, combined with excellent mutations at other sites, such as the PCR system in Example 1, amplifying the entire plasmid by PCR to obtain multiple mutations The mutants were transformed by heat shock, and the mutants were introduced into E.coli BL21 (DE3) competent cells to construct multiple mutant engineering bacteria, and the constructed multiple mutants were expressed.

[0059] 2. Determination of the product ratio and activity of wild-type strains and multiple mutants containing nitrilase

[0060] Vitality determination was carried out on the obtained nitrilase multiple mutant strains. The composition of the reaction system: 10 mL of Tris-HCl buffer solution (50 mM, pH 8.0), 10 g / L of wet bacteria, and 30 mM of phenylacetonitrile (dissolved in methanol). The reaction solution wa...

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Abstract

The invention discloses a nitrilase mutant with improved reaction specificity and application thereof, belonging to the technical field of enzyme engineering. The mutant is obtained by performing a single point mutation or multiple point mutations at the 77th, 200th, 224th or 226th position of the rice nitrilase whose amino acid sequence is shown in SEQ ID NO.1. The invention obtains a mutant with significantly improved reaction specificity by modifying the reaction non-specific rice nitrilase molecule, which is beneficial to the directional generation of the target product. The mutant Y77E / R224S / V226R constructed by the present invention catalyzes phenylacetonitrile, and the main product of the reaction is phenylacetamide, accounting for 94.8% of the product content; the constructed mutant K200R / R224W catalyzes benzylnitrile, and the main product of the reaction is phenylacetic acid, accounting for 94.8% of the product content 95.1% of the content. The nitrilase mutants with regulated reaction specificity have important industrial application potential in the green synthesis of carboxylic acid and amide compounds.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a mutant with changed specificity of nitrilase reaction and its application. Background technique [0002] Nitrile compounds are important raw materials of organic chemical industry, which are easily converted into high value-added chemicals such as amides, carboxylic acids, hydroxamic acids and ketones, and are widely used in chemical, pesticide and pharmaceutical fields. The enzymatic hydrolysis and hydration of nitrile has significant advantages such as efficient process, environmental friendliness, and high chemical, regio, and stereoselectivity, and has become an important method for the industrial synthesis of carboxylic acids and amides. [0003] It is generally believed that nitrilase can only specifically catalyze the hydrolysis of nitrile compounds to generate carboxylic acids. However, literature shows that some nitrilases have nitrile hydration activity at...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12P13/02
CPCC12N9/78C12P13/02C12Y305/05001
Inventor 郑仁朝张焱闻鹏飞郑裕国
Owner ZHEJIANG UNIV OF TECH
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