Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Fungus nitrilase mutants with improved catalytic activity and heat stability and construction method thereof

A technology of nitrilase and thermal stability, which is applied in the field of genetic engineering, can solve the problems of insufficient catalytic efficiency and poor thermal stability, and achieve the improvement of catalytic activity and thermal stability, enzyme activity and thermal stability, and broad market foreground effect

Active Publication Date: 2014-03-26
JIANGNAN UNIV +1
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the industrial production process, problems such as insufficient catalytic efficiency and poor thermal stability of the enzyme have become the main factors restricting the development and application of fungal nitrilase.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fungus nitrilase mutants with improved catalytic activity and heat stability and construction method thereof
  • Fungus nitrilase mutants with improved catalytic activity and heat stability and construction method thereof
  • Fungus nitrilase mutants with improved catalytic activity and heat stability and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The present invention uses known information to conduct bioinformatics analysis on the current fungal nitrilase gene, and establishes the important functions of the 128th isoleucine Ile and the 161st asparagine Asn near the active center for catalytic characteristics. Using a recombinant plasmid carrying the fungal nitrilase gene as a template from a methylation-capable E. coli host E. coli For DH5α, the oligonucleotide sequence with the mutation site was used as a primer to carry out saturation mutation, and the mutant plasmids at positions 128 and 161 were amplified by reverse PCR technology.

[0023] The sequences of the saturation mutation primers are as follows:

[0024]

[0025] NNN represents the mutation site, and the site was mutated into other 19 amino acids to construct a saturated mutation library.

[0026] The reverse PCR reaction system is:

[0027]

[0028] The PCR program conditions were set as follows: pre-denaturation at 94°C for 4 min; denatu...

Embodiment 2

[0030] The linear mutant plasmid recovered from rubber tapping was used Dpn Restriction endonuclease I was used for digestion and digestion, and the digestion conditions were: warm bath at 37°C for 0.5 h. The enzyme digestion reaction system is as follows:

[0031]

[0032] The digested products were detected by agarose gel electrophoresis, and the remaining products were used in subsequent experiments.

Embodiment 3

[0034] Transform the digested linear mutant plasmid directly into E. coli by heat shock at 42°C E. coli DH5α host cells were spread on LB plates containing kanamycin resistance, and cultured at 37°C for 10-12 h. Pick a single colony, insert it into LB liquid medium for culture, extract the plasmid, carry out enzyme digestion and PCR verification. The plasmids of positive clones were selected and sent to Shanghai Sangon for sequencing. Those with correct sequencing results were transformed into Escherichia coli E. coliRosetta-gami (DE3) expression host was cultured overnight at 37°C on an LB plate containing kanamycin and chloramphenicol resistance, and positive transformants were selected, which were fungal nitrilase mutants. 0.5 mM IPTG was used to induce enzyme production, and the cells were collected by centrifugation to make a bacterial suspension, and the enzyme activity of the mutant was detected, such as figure 1 shown. The mutant free cells were warmed in a wate...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides fungus nitrilase mutants with improved catalytic activity and heat stability and a construction method thereof. The mutants are obtained by adopting multiple site-saturation mutagenesis, constructing a mutual library and screening based on gibberellaintermedia CA3-1 fungus nitrilase genes. A semi-rational design method is used for obtaining the fungus nitrilase mutants and is characterized in that Ile at the site 128th and Asn at the site 161th nearby the active center of fungus nitrilase are transformed, and the obtained mutants are respectively Ile128Leu, Asn161Gln and Ile128Leu-Asn161Gln. The catalytic activity and the heat stability of the mutants provided by the invention are remarkably improved. The transformed fungus nitrilase mutants can be used for reducing the production cost and improving the production efficiency, and have relatively high application values.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a fungal nitrilase mutant with improved catalytic activity and thermal stability and a construction method thereof. Background technique [0002] Nitrilase (Nitrilase; EC 3.5.5.1) is an important biocatalyst in its super family, which can be used to hydrolyze the nitrile group (-CN) in nitrile compounds. It can exert its catalytic effect and degradation effect in pollution and other aspects, so it is often used in food additives, pharmaceutical industry, chemical production, environmental governance and other fields. In recent years, fungal nitrilase has gradually attracted attention and has become one of the very important biocatalysts. It shows great application potential in the biodegradation of industrial wastewater polluted by nitrile (Martínková, et al. Biotechnol Adv 2009, 27 (6):661-670; Malandra, et al. Appl Microbiol Biotechnol 2009, 85 (2) :277-284; Gon...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/70A23L1/305A23L29/00
CPCC12N9/78C12Y305/05001
Inventor 许正宏龚劲松李恒陆震鸣钱建瑛史劲松孙文敬周强
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com