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Cyclodextrin glucosyltransferase mutant T168A and preparation method thereof

A glucosyl and cyclodextrin technology used in the fields of genetic engineering and enzyme engineering

Active Publication Date: 2020-07-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the dismutation activity of this CGTase (about 40U / mL) is lower than that of CGTase from other sources, so it is imperative to improve the dismutation activity of this enzyme

Method used

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  • Cyclodextrin glucosyltransferase mutant T168A and preparation method thereof
  • Cyclodextrin glucosyltransferase mutant T168A and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Expression of wild-type cyclodextrin glucosyltransferase

[0030] Inoculate Cgt / pET20b(+) / BL21(DE3) (Yang Yulu, Wang Lei, Chen Sheng, etc.) from the glycerol tubes preserved in the laboratory in the early stage. The production process of β-cyclodextrin glucosyltransferase Condition optimization [J]. Biotechnology Bulletin, 2014, 8:175-181.) Grow in LB liquid medium (containing 100mg / L ampicillin) for 8 hours, and insert the seed solution into TB liquid fermentation medium according to 5% inoculum size (containing 100mg / L ampicillin). After Escherichia coli was cultured and fermented on a shaking table at 25°C for 48 hours, a certain volume of fermentation broth was centrifuged at 4°C and 12,000 rpm for 15 minutes, and the fermentation supernatant was taken, which was the crude enzyme liquid of the wild enzyme.

Embodiment 2

[0031] Example 2: Preparation and expression of cyclodextrin glucosyltransferase single mutant

[0032] (1) Single mutation preparation of cyclodextrin glucosyltransferase

[0033] According to the gene sequence of Bacillus circulans cyclodextrin glucosyltransferase, primers were designed and synthesized to introduce a single mutation, the cyclodextrin glucosyltransferase gene Cgt was subjected to site-directed mutation, and the cyclodextrin glucosyltransferase mutants were confirmed by sequencing Whether the coding gene is correct; the vector carrying the mutant gene is introduced into Escherichia coli for expression, and a single mutant cyclodextrin glucosyltransferase is obtained.

[0034] PCR amplification of the gene encoding the site-directed mutant: using the rapid PCR technique, the expression vector Cgt / pET-20b(+) carrying the gene encoding wild-type cyclodextrin glucosyltransferase was used as a template.

[0035] The site-directed mutagenesis primers for introducing ...

Embodiment 3

[0070] Example 3: Preparation and expression of cyclodextrin glucosyltransferase six mutants

[0071] (1) Preparation of six mutations of cyclodextrin glucosyltransferase

[0072] The plasmid carrying the gene encoding the mutant V6D constructed in Example 2 was used as the template for the six mutations, and the primers for site-directed mutation of S90G, T168A, T383A, and G608A designed according to Example 2 were used for rapid PCR to carry the encoding mutant. The plasmid of the V6D gene was subjected to site-directed mutation to obtain five mutants of cyclodextrin glucosyltransferase V6D / S90G / T168A / T383A / G608A. Afterwards, using the plasmid of the V6D / S90G / T168A / T383A / G608A five mutants as a template, new primers were designed to introduce the T171A mutation to construct the cyclodextrin glucosyltransferase V6D / S90G / T168A / T171A / T383A / G608A six mutants body.

[0073] The site-directed mutagenesis primers for introducing the T171A mutation were changed to:

[0074] The f...

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Abstract

The present invention discloses a cyclodextrin glucosyltransferase mutant T168A and a preparation method thereof, and belongs to the field of gene engineering and enzyme engineering. Cyclodextrin glucosyltransferase is mutated to obtain the mutant with the higher cyclodextrin glucosyltransferase disproportionation activity. The shake-flask fermentation enzyme disproportionation activity of the mutant T168A is 1.21 times that of a wild enzyme. The mutant has a certain significance for industrial production of cyclodextrin glucosyltransferase, and improves the application potential of the cyclodextrin glucosyltransferase in the food, medicine and chemical industries.

Description

technical field [0001] The invention relates to cyclodextrin glucosyltransferase mutant T168A and a preparation method thereof, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Cyclodextrin Glycosyltransferase (CGTase for short, EC2.4.1.19) is the main member of the α-amylase family (GH13). As a multifunctional enzyme, it can catalyze four different reactions, including three transfructosylation (also known as transglycoside) reactions-disproportionation, cyclization and coupling reactions, and one hydrolysis reaction. Among them, the disproportionation reaction dominates, which is to transfer the cut part of the linear oligosaccharide to another acceptor, which is an extramolecular transglycosylation reaction; the cyclization reaction is an intramolecular transglycosylation reaction. Reaction, which is a characteristic reaction of CGTase, its principle is to transfer the glycoside on the O4 or C4 of the non-reducing end o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54
CPCC12N9/1074C12Y204/01019C12N15/70C12N9/1048A23L29/06A23L33/13A23V2002/00C12N15/75C12N2800/101
Inventor 吴敬宿玲恰杜立
Owner JIANGNAN UNIV
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