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Heat-resistant beta-galactosidase mutant with transglycosylation and preparation method of mutant

A technology of galactosidase and transglycosidation activity, which is applied in the field of preparation of heat-resistant β-galactosidase mutants with transglycosidation activity, which can solve the problems of unverified catalytic changes, so as to speed up the efficiency of functional evolution and avoid Hydrolysis, the effect of increasing the amount of synthesis

Active Publication Date: 2016-09-21
JIANGNAN UNIV
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Problems solved by technology

At the same time, this method is only used in the functional modification and research of a few hydrolases, and it has not been verified whether it has any effect on the catalytic changes of other hydrolases.

Method used

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  • Heat-resistant beta-galactosidase mutant with transglycosylation and preparation method of mutant
  • Heat-resistant beta-galactosidase mutant with transglycosylation and preparation method of mutant
  • Heat-resistant beta-galactosidase mutant with transglycosylation and preparation method of mutant

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Embodiment 1

[0042] 1. Construction of E303C mutant

[0043] Through the molecular simulation of BgaB and the docking of galactose and lactose molecules with the enzyme, it is predicted that the key sites of β-galactosidase catalysis and the key amino acid sites involved in substrate binding are Glu148 and Glu303 (such as figure 1 ). Among them, Glu303 is the homologous site of nucleophile amino acid according to sequence alignment. In hydrolases with maintenance and inversion catalytic mechanisms, the catalytic residue plays a key role in the activity of the enzyme. Therefore, it is speculated that the Glu303 site may have a regulatory effect on the catalytic activity of β-galactase BgaB, so the Glu303 amino acid site was taken as the target site for research and transformation.

[0044] In this regard, the plasmid pKK223-3-bgaB containing the heat-resistant β-galactosidase gene bgab derived from Bacillus stearothermophilus was used as a template (the plasmid was provided by the Cultur...

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Abstract

The invention provides a heat-resistant beta-galactosidase mutant with transglycosylation. The mutant is obtained through the steps that beta-galactosidase (BgaB) catalyzes an amino acid site E303 to be mutated into cysteine (Cys) in a site-directed mode from original glutamic acid (Glu), and a cysteine sulfhydryl group is oxidized into -SOO<->. The invention further designs a preparation method and application of the mutant. Beta-galactosidase (BgaB) derived from bacillus stearothermophilus serves as an object, a method combining a site-directed mutant with chemical modification is adopted, the functional evolution efficiency of transglycosylation is significantly improved, the synthesis quantity of galactooligosaccharide is improved, the synthesis quantity of galactooligosaccharide is improved to 11.5% from 0% of wild-type enzyme under a medium temperature and neutral environment, limitation of reacting under an acid environment in the prior art is broken through, and meanwhile hydrolysis of a substrate or a product is avoided when galactooligosaccharide is synthesized.

Description

【Technical field】 [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a thermostable β-galactosidase mutant with transglycosidic activity, and also relates to a preparation method for a thermostable β-galactosidase mutant with transglycosidic activity. 【Background technique】 [0002] β-galactosidase (β-galactosidase) is called β-D-galactoside galactohydrolase (β-D-galactoside galacto hydrolase, EC3.2.1.23), which has two functions of catalyzing lactose hydrolysis and transglycoside . The food industry mostly uses lactose as a substrate to prepare galacto-oligosaccharides (GOS) through the transglycosidation of β-galactosidase. The β-galactosidase BgaB derived from Bacillus stearothermophilus involved in the present invention belongs to the GH42 family and is a heat-resistant enzyme with industrial application potential. Hydrolysis and transglycoside catalyzed reactions. However, due to the weak transglyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N15/56C12N15/70C12P19/14
CPCC12N9/2471C12N15/70C12N2800/101C12P19/14C12Y302/01023
Inventor 陈海琴董艺凝陈卫赵建新陈永泉张灏
Owner JIANGNAN UNIV
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