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Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof

A technology of glucosidase and mutants, applied in the field of bioengineering, can solve the problems of difficult enzyme purification and low catalytic efficiency

Active Publication Date: 2015-06-24
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 it is difficult to purify the artificially extracted and expressed enzymes. If rational molecular design can be carried out through homology modeling and other means, the endogenous D The rationalization of catalytic activity, stability, substrate specificity, heat resistance and acid and alkali resistance of type β-glucosidase protein will make this type of enzyme have greater application prospects and realize huge industrial economic value

Method used

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  • Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof
  • Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof
  • Beta-glucosidase D mutant as well as expression plasmid and recombinant bacteria thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of mutant expression plasmid and obtaining of recombinant E. coli mutant expression strain

[0028] 1. Construction of mutant expression plasmid

[0029] Gene in vitro cloning and site-directed mutation of D-type β-glucosidase by means of genetic engineering, with

[0030] The overall process is as follows:

[0031] RNA was extracted from the salivary glands and intestinal tissues of Taiwanese termites raised in the laboratory, and cDNA was obtained through reverse transcription.

[0032] The β-glucosidase gene of Taiwanese termite was found in the NCBI gene database ( JN565080 ), according to the sequence homology, design amplification primers, use the termite cDNA as a template for amplification, and add restriction enzymes Hin d III and Xho The restriction site of I, the PCR amplified product is inserted between the corresponding multiple cloning sites on the vector pET-28a (Novagen), and transformed into Ecoli .DH5α (Novagen company) in competent ...

Embodiment 2

[0057] Example 2: Determination of D-type β-glucosidase activity before and after mutation

[0058] According to the method in Example 1, the original Escherichia coli pET-28a-Glu1D and other mutant strains were induced to express, and the enzyme activity was measured, and the results were as follows figure 2 Shown by the attached figure 2 It can be seen that the enzyme activity of the strain after the mutation is 94.6, 92.9, 100.3, 71.4, 69.9 times higher than that before the mutation, respectively.

[0059] The activity of the enzyme after the mutation of the present invention is greatly improved compared with that before the mutation. The results show that mutation of amino acids at key positions can change the activity and other properties of the enzyme. This strategy can be widely used to improve the properties of enzymes, accelerate the application of enzymes in industrial production, and has important social significance.

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Abstract

The invention relates to a beta-glucosidase D mutant as well as an expression plasmid and recombinant bacteria thereof and in particular relates to a beta-glucosidase D mutant with obviously improved enzymatic activities as well as an expression plasmid and recombinant bacteria thereof, belonging to the technical field of bioengineering. Site-specific mutation is carried out on beta-glucosidase derived from Formosan termites to change tyrosine Y in the 182 bit to tryptophan W and leucine L respectively, change methionine M in the 194 bit to leucine L and phenylalanine F respectively and change aspartic acid D in the 244 bit to histidine H, wherein tryptophan W, leucine L, leucine L, phenylalanine F and histidine H are respectively expressed as Glu1D-Y182W, Glu1D-Y182L, Glu1D-M194L, Glu1D-M194F and Glu1D-D244H. The enzymatic activities of the mutant are respectively improved by 94.6 times, 92.9 times, 100.3 times, 71.4 times and 69.9 times compared with the enzymatic activities before mutation. The obtained beta-glucosidase mutant has substantially improved enzymatic activities, provides a favourable foundation for industrial application, can further meet the requirements of social production and has extensive application prospects.

Description

Technical field [0001] The invention relates to a D-type β-glucosidase mutant and its expression plasmid and recombinant bacteria, in particular to a D-type β-glucosidase mutant with significantly improved enzyme activity, its expression plasmid and recombinant bacteria, It belongs to the field of biological engineering technology. Background technique [0002] D-type β-glucosidase (β-D-glucopyranoside hydrolase, E.C. 3.2.1.21) is a class of enzymes that can hydrolyze the glycosidic bonds of glycosides and oligosaccharides and release non-reducing terminal glucose residues. These enzymes are ubiquitous in all fields of life, and play a variety of functions and roles in archaea, eubacteria and eukaryotes. Including biomass conversion in microorganisms, degradation of animal body glycolipids and exogenous glycosides, lignification and catabolism of cell wall oligosaccharides, defense, phytohormone binding and conjugate activation, odor release in plants, and plant-microbes and pla...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21
CPCC12Y302/01021
Inventor 施海峰冯婷婷刘海涛王继峰吴黎明周阳
Owner JIANGSU UNIV
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