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Nitrilase mutant, engineering bacterium and application of nitrilase mutant

A technology of nitrilase and mutant, applied in the directions of hydrolase, application, genetic engineering, etc.

Active Publication Date: 2022-04-05
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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  • Nitrilase mutant, engineering bacterium and application of nitrilase mutant
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  • Nitrilase mutant, engineering bacterium and application of nitrilase mutant

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Embodiment 1

[0023] The construction of embodiment 1 nitrilase mutant

[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, it was determined that its catalytic triplet was 196Cys-71Glu-162Lys, and then through rational design analysis, the 145th cysteine, the 200th lysine, the 224th arginine, and the 229th alanine Amino acid and asparagine at position 246 were subjected to site-directed saturation mutation, and a mutant with significantly improved reaction specificity was constructed, specifically:

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

Embodiment 2

[0050] Example 2 Nitrilase Mutant C145N, K200R / R224W, A229P, N246V Reaction Specificity and Activity Determination

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

[0052] The reaction system (20mL) consists of: 20mL Tris-HCl buffer solution (50mM, pH 8.0), 0.1g of wet thallus of the nitrilase mutant prepared by the method of Example 1, 0.9mL of methanol, 100mM of phenylacetonitrile (benzylnitrile first soluble in methanol). The reaction solution was reacted at 30° C., 180 rpm for 30 minutes. Take 1 mL of sample, add 20 μL of 2M HCl to terminate ...

Embodiment 3

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

[0058] The results of the single point mutation in Example 1 were analyzed, and it was found that the 200th, 224th, and 229th point mutations can change the specificity of the reaction so that it tends to generate carboxylic acid, and the hydrolysis activity is better retained or improved. The hydration activity was inhibited, and the mutant K200R / R224W / A229P was obtained by superposition mutation, specifically:

[0059] Using the K200R / R224W mutant plasmid constructed in Example 2 as a template, the primers at position 229 (Table 1Ala229-For, Ala229-Rev) were used for PCR amplification of the whole plasmid. The PCR system and reaction conditions were the same as in step 1, and Example 1 was used. Methods The plasmid containing the nitrilase mutant K200R / R224W / A229P was constructed.

[0060] Cultivate and obtain wet thalline cells capable of measuring carboxylic acid ratio and activity by the meth...

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Abstract

The invention discloses a nitrilase mutant, an engineering bacterium and application of the nitrilase mutant and the engineering bacterium. The amino acid sequence of the nitrilase mutant is shown as SEQ ID NO.5. The main product generated by catalyzing benzyl cyanide by the mutant K200R / R224W / A229P constructed by the invention is phenylacetic acid which accounts for 96.3% of the product content, the nitrile hydration activity is reduced by 82.2% compared with that of K200R / R224W, and directional generation of the target product phenylacetic acid is facilitated. The nitrilase can be applied to green industrial catalytic synthesis of amide and carboxylic acid through reaction specificity regulation and control, and has important significance.

Description

(1) Technical field [0001] The invention relates to a nitrilase mutant with improved nitrile hydrolysis activity specificity 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. Nitrilase can catalyze the hydrolysis of nitrile compounds to generate carboxylic acids, and has unique advantages such as chemoselectivity, stereoselectivity and regioselectivity, and plays an important role in the conversion of nitrile compounds. In addition to catalyzing the hydrolysis of nitrile compounds to carboxylic acids, some nitrilases also have nitrile hydration activity. 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 asparagine and aspartic acid...

Claims

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Application Information

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Patent Type & Authority Applications(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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