Nitrilase mutant with improved nitrile hydration activity specificity and application thereof

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

Active Publication Date: 2021-04-09
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Further research found that the hydration activity of Y77E / R224S / V226R was only 74.35% of the wild type

Method used

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  • Nitrilase mutant with improved nitrile hydration activity specificity and application thereof
  • Nitrilase mutant with improved nitrile hydration activity specificity and application thereof
  • Nitrilase mutant with improved nitrile hydration activity specificity and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The construction of embodiment 1 nitrilase mutant recombinant bacteria

[0029] 1. Construction of mutants

[0030] 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 87th alanine, the 136th isoleucine, the 224th arginine, and the 226th Valine was subjected to site-directed saturation mutation, and a mutant with significantly increased amide content was constructed, specifically:

[0031] (1) Single point mutation: insert the rice nitrilase (OsNIT, GenBank accession number: AB027054) gene (amino acid sequence SEQ ID NO.1, nucleotide sequence is SEQ ID NO.2) into the BamH I of the pET-28b vector With the Hind III site, a recombinant plasmid was constructed. Usin...

Embodiment 2

[0052] Embodiment 2 nitrilase mutant A87M, I136Q amide content and activity determination

[0053] The reaction system consists of: 20mL Tris-HCl buffer solution (50mM, pH 8.0), 0.1g of wet thallus of nitrilase mutants A87M and I136Q prepared by the method in Example 1, methanol 0.9mL, phenylacetonitrile 100mM (phenylacetonitrile 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 the reaction, centrifuge at 12,000 rpm for 1 min, and take the supernatant. The concentration and ratio of phenylacetic acid and phenylacetamide in the product were analyzed by high performance liquid chromatography HPLC described in Example 1, and the enzyme activity was calculated. Under the same conditions, the enzyme activity of the nitrilase mutant wet cells was tested.

[0054] Among them, while the hydration activity of the A87M (SEQ ID NO.4) mutant was 107% of the parent, the hydrolysis activ...

Embodiment 3

[0057] Embodiment 3 Construction of nitrilase multiple mutant R224S / V226R / I136Q / A87M

[0058] Using the above method to analyze the results of single-point mutations, it was found that the mutations at the 136th and 87th positions could change the specificity of the reaction and tend to generate amides, the hydration activity was well preserved, and the hydrolysis activity was inhibited. R224S , V226R on the basis of further mutation transformation, the specific steps are as follows:

[0059] Using the R224S mutant plasmid as a template, use the V226R site-directed mutagenesis primers (Table 1 Val226-For and Val226-Rev) to carry out PCR amplification of the whole plasmid. The PCR system and amplification conditions are the same as step 1. Enzyme mutant R224S / V226R plasmid (amino acid sequence SEQ ID NO.3); then use the mutant R224S / V226R plasmid as a template, and use I136Q site-directed mutation primers (Table 1Ile136-For and Ile136-Rev) to carry out PCR amplification of the ...

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Abstract

The invention discloses a nitrilase mutant with improved nitrilase hydration activity specificity and application thereof. The mutant is obtained by carrying out single mutation or multiple mutation on the 87th site, the 136th site, the 224th site and the 226th site of an amino acid sequence shown in SEQ ID NO.1. When the mutant R224S/V226R/I136Q/A87M constructed in the invention is used for catalyzing phenylacetonitrile, a reaction main product is phenylacetamide, the content of amide reaches 91.2%, and the nitrile hydration activity is 113.3% of that of OsNIT. When the phenylacetonitrile is catalyzed by the wild nitrilase OsNIT, the content of the phenylacetamide is 50.6%, and meanwhile, the nitrile hydration activity of the OsNIT is 100%. The amide content and the hydration activity of the R224S/V226R/I136Q/A87M are 1.53 times and 1.13 times of those of the wild nitrilase respectively. The reaction specificity of the nitrilase is regulated and controlled, so that the nitrilase can be applied to amide green industrial catalytic synthesis, and important significance is achieved.

Description

[0001] (1) Technical field [0002] The invention relates to a nitrilase mutant with improved specificity of nitrile hydration activity and its application in catalyzing the synthesis of phenylacetamide from benzyl nitrile. [0003] (2) Background technology [0004] 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70C12N1/21C12P13/02C12R1/19
CPCC12N9/78C12N15/70C12N1/20C12P13/02C12Y305/05001
Inventor 郑仁朝闻鹏飞汤晓玲郑裕国
Owner ZHEJIANG UNIV OF TECH
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