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

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

Active Publication Date: 2022-05-24
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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  • A kind of nitrilase mutant with improved nitrile hydration activity specificity and application thereof
  • A kind of nitrilase mutant with improved nitrile hydration activity specificity and application thereof
  • A kind of nitrilase mutant with improved nitrile hydration activity specificity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of nitrilase mutant recombinant bacteria

[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 87th alanine, the 136th isoleucine, the 224th arginine, and the 226th position were selected. Valine was subjected to site-directed saturation mutation to construct mutants with significantly increased amide content, specifically:

[0026] (1) Single point mutation: The rice nitrilase (OsNIT, GenBank accession number: AB027054) gene (amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) was inserted into the BamH I of the pET-28b vector With the Hind III site, construct the recombinant plasmid. Us...

Embodiment 2

[0047] Example 2 Determination of nitrilase mutant A87M, I136Q amide content and activity

[0048] The reaction system was composed of: 20 mL Tris-HCl buffer solution (50 mM, pH 8.0), 0.1 g of nitrilase mutant A87M and I136Q 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 2M HCl to stop the reaction, centrifuging at 12,000 rpm for 1 min, and taking the supernatant. The concentration and ratio of phenylacetic acid and phenylacetamide in the product were analyzed by high performance liquid chromatography (HPLC) as described in Example 1, and the enzymatic activity was calculated. The enzyme activity of the nitrilase mutant wet cells was tested under the same conditions.

[0049] Among them, the A87M (SEQ ID NO. 4) mutant has a hydration activity of 107% of the parent, while the hydroly...

Embodiment 3

[0052] Example 3 Construction of nitrilase multiple mutants R224S / V226R / I136Q / A87M

[0053] Using the above method to analyze the results of single-point mutation, it was found that the mutations at the 136th and 87th positions can change the specificity of the reaction and make it tend to generate amides, the hydration activity is well preserved, and the hydrolysis activity is inhibited. R224S , V226R based on further mutation transformation, the specific steps are as follows:

[0054] Taking the R224S mutant plasmid as a template, the V226R site-directed mutagenesis primers (Table 1 Val226-For and Val226-Rev) were used to carry out PCR amplification of the whole plasmid. The PCR system and amplification conditions were the same as in step 1, and the method of Example 1 was used to construct a nitrile hydrolysis solution. The enzyme mutant R224S / V226R plasmid (amino acid sequence SEQ ID NO. 3); then the mutant R224S / V226R plasmid was used as a template, and the I136Q site-dir...

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Abstract

The invention discloses a nitrilase mutant with improved specificity of nitrile hydration activity and its application. The mutant is the 87th, 136th, 224th, 224th, The 226th position is obtained by single or multiple mutations. When the mutant R224S / V226R / I136Q / A87M constructed by the present invention catalyzes phenylacetonitrile, the main product of the reaction is phenylacetamide, the amide content reaches 91.2%, and the nitrile hydration activity is 113.3% of OsNIT. When the wild-type nitrilase OsNIT catalyzes phenylacetonitrile, the content of phenylacetamide is 50.6%, and the hydration activity of OsNIT nitrile is recorded as 100%. The amide content and hydration activity of R224S / V226R / I136Q / A87M were 1.53 times and 1.13 times that of the wild type, respectively. 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.

Description

(1) Technical field [0001] The present invention relates to a nitrilase mutant with improved specificity of nitrile hydration activity and its application in catalyzing phenylacetonitrile to synthesize phenylacetamide. (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-cy...

Claims

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Application Information

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Patent Type & Authority Patents(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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