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Pseudomonas putida nitrilase mutant obtained by site-directed mutation and its construction method

A nitrilase and activity technology, applied in the field of genetic engineering, can solve the problems of low specific enzyme activity, narrow substrate spectrum, poor stability, etc., and achieve the effect of high enzyme activity and broad application prospects

Active Publication Date: 2020-08-25
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many limitations in the application of nitrilase, such as low specific enzyme activity, poor stability, narrow substrate spectrum, etc.

Method used

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  • Pseudomonas putida nitrilase mutant obtained by site-directed mutation and its construction method
  • Pseudomonas putida nitrilase mutant obtained by site-directed mutation and its construction method
  • Pseudomonas putida nitrilase mutant obtained by site-directed mutation and its construction method

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0018] The construction method of the nitrilase mutant strain that above-mentioned enzyme activity improves comprises the following steps:

[0019] 1) Pseudomonas putida ( Pseudomonas putida CGMCC3830) Construction of Nitrilase Engineering Bacteria

[0020] For the Pseudomonas putida nitrilase gene, the target gene was amplified by upstream primers and downstream primers:

[0021] Upstream primer: 5'-CCG GAATTC ATGGTTACGTACACGAATAAGTTCA-3', the underlined base is the restriction endonuclease EcoRI recognition site;

[0022] Downstream primer: 5'-CCC AAGCTT GACCGGGGACTTCCAAGCTATACGTT-3', the underlined base is the recognition site of restriction endonuclease HindⅢ;

[0023] Using the Pseudomonas putida genome as a template, the upstream and downstream primers participated in the PCR reaction. The PCR conditions were pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 90 s, and 30 cycles ; Final extension a...

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Abstract

Belonging to the field of gene engineering, the invention provides a high enzyme activity nitrilase mutant strain and a preparation method thereof. According to the invention, Pseudomonas putida CGMCC3830 nitrilase is adopted as the template, and then saturated site directed mutagenesis is carried out on a Pseudomonas putida nitrilase sequence by a molecular biological means so as to obtain two enzyme activity increased positive transformants Asn40Gly and Phe50Trp, and on the basis, combination mutation is conducted to obtain a combined mutant strain Asn40Gly-Phe50Trp. Under transformation conditions, the enzyme activities are all enhanced, and the thermal stability is also improved. By the strategy, the transformation ability of nitrilase can be greatly improved, so that the nitrilase mutant strain can be applied in pharmaceutical intermediates, food additives and environmental governance aspects, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a mutant strain of Pseudomonas putida nitrilase with improved enzyme activity and a construction method thereof. Background technique [0002] Nitrilase is a kind of catalyst that can hydrolyze nitrile compounds into carboxylic acid and ammonia in one step. Its stereoselectivity and regioselectivity can be used to solve the problem of low efficiency of chiral catalysis and regiocatalysis in chemical processes. Nitrilase has a wide range of sources, mild reaction conditions, high specificity and strong selectivity, and has potential application value in the field of organic synthesis. However, there are still many limitations in the application of nitrilase, such as low specific enzyme activity, poor stability, and narrow substrate spectrum. Therefore, obtaining a nitrilase with improved enzyme activity has important industrial application value. [0003] In this s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70
CPCC12N9/78C12Y305/05001
Inventor 龚劲松熊雷许正宏李恒史劲松孙文敬周强
Owner JIANGNAN UNIV