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A kind of nitrilase mutant with improved specificity of nitrile hydrolysis activity and application thereof

A kind of nitrilase, mutant technology, applied in the direction of hydrolase, application, enzyme, etc.

Active Publication Date: 2022-02-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Zhang Yan et al. modified rice (Oryza sativa) nitrilase OsNIT, when the mutant K200R / R224W used phenylacetonitrile as the substrate, the carboxylic acid content reached 95.1% (CN111321132A), but the nitrile hydration activity of the mutant K200R / R224W was still wild type 22.74% of

Method used

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  • A kind of nitrilase mutant with improved specificity of nitrile hydrolysis activity and application thereof
  • A kind of nitrilase mutant with improved specificity of nitrile hydrolysis activity and application thereof
  • A kind of nitrilase mutant with improved specificity of nitrile hydrolysis activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of nitrilase mutants

[0024] 1. Construction of mutants

[0025] When rice nitrilase (OsNIT, GenBank accession number: AB027054, amino acid sequence SEQ ID NO. 1) catalyzes the reaction of phenylacetonitrile, the ratio of amide to carboxylic acid in the product is close to 1:1. Through bioinformatics analysis, the catalytic triplet was determined to be 196Cys-71Glu-162Lys, and then through rational design analysis, the 145th cysteine, the 200th lysine, the 224th arginine, and the 229th clan Site-directed saturation mutation of amino acid and asparagine at position 246 to construct a mutant with significantly improved reaction specificity, specifically:

[0026] The rice nitrilase (OsNIT, GenBank accession number: AB027054) gene (amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) was inserted between the BamH I and Hind III sites of the pET-28b vector to construct recombinant plasmids. Taking the recombinant plasmid as a template...

Embodiment 2

[0050] Example 2 Determination of reaction specificity and activity of nitrilase mutants C145N, K200R / R224W, A229P, N246V

[0051] The plasmid of the K200R mutant obtained in Example 1 was used as a template, and the R224W site-directed mutagenesis primer (Table 1 Lys200-For, Lys200-Rev) was used to carry out PCR amplification of the whole plasmid. The PCR system and reaction conditions were the same as in Step 1, and the method of Example 1 was adopted. A plasmid of mutant K200R / R224W (amino acid sequence shown in SEQ ID NO. 3) was constructed, and wet cells were prepared according to the method of Example 1.

[0052] The reaction system (20 mL) was composed of: 20 mL Tris-HCl buffer solution (50 mM, pH 8.0), 0.1 g of nitrilase mutant wet cells prepared by the method of Example 1, 0.9 mL of methanol, 100 mM of phenylacetonitrile (phenylacetonitrile first). soluble in methanol). The reaction solution was reacted at 30° C. and 180 rpm for 30 min. Sampling 1 mL, adding 20 μL 2...

Embodiment 3

[0057] Example 3 Construction of the nitrilase multiple mutant K200R / R224W / A229P.

[0058] The single-point mutation results of Example 1 were analyzed, and it was found that the 200th, 224th, and 229th site mutations could change the reaction specificity to make it tend to generate carboxylic acid, and the hydrolysis activity was better preserved or improved, The hydration activity was inhibited, and the mutant K200R / R224W / A229P was obtained by superimposing mutations, specifically:

[0059] Take the K200R / R224W mutant plasmid constructed in Example 2 as a template, adopt 229 site primers (Table 1Ala229-For, Ala229-Rev) to carry out PCR amplification of the whole plasmid, PCR system and reaction conditions are the same as step 1, using Example 1 Methods A plasmid containing nitrilase mutant K200R / R224W / A229P was constructed.

[0060] According to the method of Example 1, wet bacterial cells capable of measuring the ratio of carboxylic acid and viability were obtained by cult...

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Abstract

The invention discloses a nitrilase mutant with improved specificity of nitrile hydrolysis activity and its application. The mutant is the 200th, 224th, 229th, 229th, 229th, 229th, The 246th position is obtained by single or multiple mutations. The main product produced by the mutants C145N, K200R / R224W / A229P, and K200R / R224W / N246V catalyzed by the present invention is phenylacetic acid, which accounts for 96.5%, 96.3% and 98.8% of the product content respectively. The ratio of nitrile hydration activity to K200R / R224W decreased by 88.3%, 82.2% and 94.3% respectively, which is conducive to the directional formation of the target product phenylacetic acid. It is of great significance to regulate the reaction specificity of nitrilase so that it can be applied to the green industrial catalytic synthesis of amides and carboxylic acids.

Description

(1) Technical field [0001] The present invention relates to a nitrilase mutant with improved specificity of nitrile hydrolysis activity and its application in synthesizing phenylacetic acid. (2) Background technology [0002] Nitrile compounds are a class of organic chemical raw materials containing cyano groups, which are widely used in chemical, pharmaceutical, pesticide and material industries. Nitrilases can catalyze the hydrolysis of nitrile compounds to generate carboxylic acids, and have unique advantages such as chemoselectivity, stereoselectivity and regioselectivity, and play an important role in the transformation of nitrile compounds. Some nitrilases not only catalyze the hydrolysis of nitrile compounds to form carboxylic acids, but also have nitrile hydration activities. For example, Piotrowski et al. found that Arabidopsis nitrilase has both nitrile hydrolysis and nitrile hydration activities, and can catalyze the conversion of β-cyano-L-alanine into asparagin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P7/40C12R1/19
CPCC12N9/78C12N15/70C12N1/20C12P7/40C12Y305/05001
Inventor 郑仁朝汤晓玲闻鹏飞郑裕国
Owner ZHEJIANG UNIV OF TECH
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