Application of heat-resistant beta-glucosidase and mutants thereof

A technology of glucosidase and heat resistance, applied in the direction of glycosylase, enzyme, hydrolase, etc., can solve the problem of low specificity, achieve high conversion efficiency, less reaction by-products, and high purity

Inactive Publication Date: 2014-02-19
NANJING FIRST HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these enzymes have a wide range of substrates (reported substrates: cellobiose, lactose, salicin, arbutin, alkyl glycosides, p-nitrophenol-β-D-glucoside and p-nitrophenol -β-D-fructoside, p-nitrophenol-β-D-galactoside, daidzin, genistin, malonyl daidzein, malonyl genistin, laminaribiose and sophorose), Specificity of the usual hydrolyzed substrate quercetin glycosides (quercetin-3-O-glucoside, quercetin-4'-O-glucoside and quercetin-3,4'-O-glucoside) not tall

Method used

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  • Application of heat-resistant beta-glucosidase and mutants thereof
  • Application of heat-resistant beta-glucosidase and mutants thereof
  • Application of heat-resistant beta-glucosidase and mutants thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] According to the β-glucosidase gene sequence of family 1 of Thermotoga (Thermotiga), sequence number: X74163), design mutation primers, specifically

[0026] 5'-AGTTTTCAACAACACCTATTTCGAACCTGCGAGT-3' (SEQ ID NO. 4) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO. 5);

[0027]The recombinant plasmid pET-20b-bglA (J Ind Microbiol Biotechnol, 2009, 36:1401–1408) containing the wild-type β-glucosidase gene of Thermotoga was selected as a template for inverse PCR amplification to obtain β-glucosidase The nucleotide fragment of the enzyme mutant gene is phosphorylated, connected, transformed, and the plasmid is extracted to obtain a recombinant plasmid containing the G224T nucleotide fragment, and the host is further transformed to express it, and the β-glucoside of the present invention is obtained The amino acid sequence of the enzyme mutant G224T is shown in SEQ ID NO.1.

Embodiment 2

[0029] It is basically the same as Example 1, except that the primers used are: 5'-AGTTTTCAATAGTGGATATTTCGAACCTGCGAGT-3' (SEQ ID NO.6) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO.7), to obtain The recombinant plasmid of the nucleotide fragment is further transformed into a host to express it, thereby obtaining the β-glucosidase mutant N223S of the present invention; its amino acid sequence is shown in SEQ ID NO.2.

Embodiment 3

[0031] It is basically the same as Example 1, except that the primers used are: 5'-AGTTTTCAATAGTACCTATTTCGAACCTGCGAGT-3' (SEQ ID NO.8) and 5'-ATTCCGATCTTTCCATCTTTCACG-3' (SEQ ID NO.9), to obtain The recombinant plasmid of / G224T nucleotide fragment is further transformed into a host to express it, thereby obtaining the β-glucosidase mutant N223S / G224T of the present invention; its amino acid sequence is shown in SEQ ID NO.3.

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Abstract

The invention discloses application of heat-resistant beta-glucosidase and mutants thereof. The mutants of the heat-resistant beta-glucosidase G224T, N223S and N223S / G224T have the amino acid sequences shown in SEQ: ID NO. 1, SEQ: ID NO. 2 and SEQ: ID NO. 3; the heat-resistant beta-glucosidase and the mutants thereof can be used for preparing quercetin through enzymolysis of quercetin glycoside. The preparation of the quercetin through the heat-resistant beta-glucosidase and the mutants thereof has the advantages of high conversion efficiency, few reaction byproducts, easy separation and purification of the product, less chemical pollution, and high yield and high purity of the obtained quercetin product, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a heat-resistant β-glucosidase mutant N223S, G224T and N223S / G224T, and the enzyme and its mutant in the enzymatic hydrolysis of quercetin glycoside to prepare quercetin application. technical background [0002] Carbohydrates are the most predominant compounds present in biomass, with an estimated 6 billion tons produced annually. In order to utilize these natural raw materials, microorganisms produce a variety of glycoside hydrolases that can hydrolyze and modify carbohydrates (such as aglycone including aromatic groups and alkane groups, sugar groups including glucose, lactose, fructose and xylose), These enzymes can catalyze the hydrolysis of the β-D-glucosidic bond bound to the non-reducing terminal, and at the same time release the ligand and glucosome, and are widely used as specific catalysts in food processing, feed industry, pulp and paper industry, starch, textile , and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12P17/06C12R1/01
CPCC12N9/2445C12P17/06C12Y302/01021
Inventor 曹志刚薛业敏林宇菲孙慧慧
Owner NANJING FIRST HOSPITAL
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