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A β-glucosidase with increased resistance to trypsin and pepsin

A technology of glucosidase and pepsin, applied in the direction of enzyme, hydrolase, glycosylase, etc.

Active Publication Date: 2021-07-27
GUANGDONG GENUIZYMES ANIMAL HEALTH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, β-glucosidase with improved pepsin resistance has not been reported yet.

Method used

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  • A β-glucosidase with increased resistance to trypsin and pepsin
  • A β-glucosidase with increased resistance to trypsin and pepsin
  • A β-glucosidase with increased resistance to trypsin and pepsin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the synthesis of β-glucosidase gene

[0029] The present invention adopts the gene of wild-type β-glucosidase derived from Trichoderma viride (GenBank registration number is FJ882071.1), which is synthesized by Jinweizhi Company (other commercial companies with full gene synthesis can also complete it).

Embodiment 2

[0030] Embodiment 2: β-glucosidase (bgl1) and cloning carrier Tao x +PgHT+PB connection

[0031] 1. Digest the target gene of mbgl1 and the cloning vector Taox+PgHT+PB with restriction endonucleases EcoRI and SpeI / XbaI at 37°C for 10 min respectively. The digestion conditions are as follows:

[0032]

[0033] 2. After the digested products were subjected to 1% agarose gel electrophoresis, the two target fragments were respectively recovered and ligated with T4DNA ligase. The ligation system was as follows:

[0034] bgl1 digestion product 0.6pmol Cloning Vector Restriction Products 0.08pmol T4 DNAse 1μl 10× buffer 1μl Total 10μl

[0035] Use ligase to ligate at 16°C for 12 hours, transform the ligation product into DH5a competent cells and amplify, extract the plasmid with a plasmid extraction kit, digest with EcoRI and PstI, and then run electrophoresis. The results show two bands of 7.0kb and 3.8kb. It showed that the connectio...

Embodiment 3

[0036] Embodiment 3: gene fragment Paox+SS1 is connected with cloning vector M+Taox+PgHT+PB

[0037] 1. The gene fragment Paox+SS1 was transferred from the cloning vector Paox+SS1+PB preserved in this research institute, and obtained by double digestion with EcoRI and SpeI endonucleases, purification and recovery;

[0038] 2. The cloning vector M+Taox+PgHT+PB was obtained from Example 2, and the connection method between the gene fragment Paox+SS1 and the cloning vector M+Taox+PgHT+PB was the same as in Example 2.

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Abstract

The invention discloses a β-glucosidase with improved resistance to trypsin and pepsin and application thereof. The present invention transforms key amino acid residues in wild-type β-glucosidase molecules through protein engineering technology, and provides a β-glucosidase mutant with improved resistance to trypsin and pepsin. The mutant β-glucosidase (mBGL1) acting on cellobiose and short-chain cellooligosaccharides described in the present invention has improved resistance to trypsin and pepsin than wild-type BGL1, and other enzymatic properties are similar to wild-type BGL1. Enzymes are basically the same.

Description

technical field [0001] The present invention relates to beta-glucosidases, and in particular to beta-glucosidases with increased resistance to trypsin and pepsin. Background technique [0002] β-glucosidase (β-D-Glucosidase, EC3.2.1.21, referred to as BGL1), also known as glucoside hydrolase, can hydrolyze cellobiose and short-chain cellooligosaccharides to generate glucose, and decompose cellobiose and fiber The feedback inhibition of oligosaccharides on endoglucanase and cellobiohydrolase can also enzymolyze the aroma precursors in fruits and tea to enhance the aroma. β-glucosidase belongs to hydrolytic enzymes and is one of the important components of cellulase system. [0003] β-glucosidase has important physiological functions in the degradation of glycogen in humans and the carbohydrate metabolism of animals, plants and microorganisms. In recent years, it has shown important application prospects in the food industry: β-glucosidase can hydrolyze the flavor precursors...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2445C12N15/815C12Y302/01021
Inventor 姚冬生汪浩谢春芳刘大岭
Owner GUANGDONG GENUIZYMES ANIMAL HEALTH CO LTD