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A kind of c-type β-glucosidase mutant and its expression plasmid and recombinant bacteria

A technology of glucosidase and mutants, which is applied in the field of C-type β-glucosidase mutants and their expression plasmids and recombinant bacteria, which can solve the problems of difficult enzyme purification and low catalytic efficiency

Inactive Publication Date: 2018-01-16
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that the catalytic efficiency of this type of cellulolytic enzymes is low, and the purification of artificially extracted and expressed enzymes is relatively difficult. If rational molecular design can be carried out through homology modeling and other means, the endogenous β -Rationalization of the catalytic activity, stability, substrate specificity, heat resistance and acid and alkali resistance of glucosidase protein will make this kind of enzyme have greater application prospects and realize huge industrial economic value

Method used

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  • A kind of c-type β-glucosidase mutant and its expression plasmid and recombinant bacteria
  • A kind of c-type β-glucosidase mutant and its expression plasmid and recombinant bacteria
  • A kind of c-type β-glucosidase mutant and its expression plasmid and recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of mutant expression plasmids and acquisition of recombinant Escherichia coli

[0029] 1. Construction of mutant expression plasmids

[0030] According to protein structure prediction and homologous sequence analysis, a series of mutation sites were found, and point mutations were carried out on these amino acid sites to obtain a series of mutant enzymes. After protein expression and verification, it was found that the catalytic activity of several mutant enzymes was significantly enhanced. The build process is as follows:

[0031] (1) RNA was extracted from the salivary glands and intestinal tissues of domestic termites reared in the laboratory, and cDNA was obtained after reverse transcription.

[0032] (2) The β-glucosidase gene ( JN565079 ), according to the sequence homology, design amplification primers, amplify the termite cDNA obtained in step (1), after PCR amplification, insert the amplified product into the corresponding polyclonal...

Embodiment 2

[0054] Example 2: Activity determination of C-type β-glucosidase before and after mutation

[0055] According to the method in Example 1, the original Escherichia coli pET-28a-Glu1C and other mutant strains were induced to express, and the enzyme activity was measured, and the results were as follows image 3 shown, by attaching image 3 It can be seen that the enzyme activity of the strain after the mutation was increased by 5.2 times, 2.05 times, 2.9 times, and 2.1 times, respectively, compared with that before the mutation.

[0056] The activity of the enzyme after the mutation described in the present invention is greatly improved compared with that before the mutation. The results show that mutating amino acids at key sites can change the activity and other properties of the enzyme. This strategy can be widely used in the improvement of enzymatic properties and accelerates the application of enzymes in industrial production, which has important social significance.

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Abstract

The invention relates to a C-type β-glucosidase mutant and its expression plasmid and recombinant bacteria, in particular to a C-type β-glucosidase mutant with significantly improved enzyme activity and a construction method thereof, belonging to biological engineering Technical field; The present invention carries out site-directed mutation on the β-glucosidase derived from Taiwan termites, and changes the lysine K at the 170th position into serine S and tryptophan W respectively, and the phenylalanine F at the 182nd position is methionine M, the amino acid histidine H at the 252nd position becomes asparagine N, and is respectively expressed as Glu1C-K170S, Glu1C-K170W, Glu1C-F182M, Glu1C-H252N; the obtained mutant of the present invention Enzyme activity has improved respectively 5.2 times, 2.05 times, 2.9 times, 2.1 times compared with before mutation; The application of biocatalytic technology provides a favorable basis for industrial application, can better meet the requirements of social production, and has broad market prospects.

Description

technical field [0001] The invention relates to a C-type β-glucosidase mutant and its expression plasmid and recombinant bacteria, in particular to a C-type β-glucosidase mutant with significantly improved enzyme activity and its construction method, which belongs to the biological [0002] field of physical engineering technology. Background technique [0003] Type C β -glucosidase ( β -C-glucopyranoside hydrolases, E.C. 3.2.1.21) are a class of enzymes that hydrolyze the glycosidic bonds of glycosides and oligosaccharides and release non-reducing terminal glucose residues. These enzymes are ubiquitously present in all domains of life, performing multiple functions and roles in archaea, eubacteria, and eukaryotes. Including biomass turnover in microorganisms, degradation of glycolipids and exogenous glycosides in animals, lignification and catabolism of cell wall oligosaccharides, defense, phytohormone binding conjugate activation, odor release in plants, and plant-micro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/42C12N15/56C12N1/21C12R1/19
Inventor 王继峰吴黎明冯婷婷刘海涛周阳施海峰
Owner JIANGSU UNIV
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