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A β-glucosidase with increased resistance to trypsin

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

Active Publication Date: 2022-06-21
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 trypsin resistance has not been reported yet.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Synthesis of β-glucosidase gene

[0028] The present invention adopts the wild-type β-glucosidase gene (GenBank registration number: FJ882071.1) derived from Trichoderma viride, which is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (other commercial companies with full gene synthesis can also complete it).

Embodiment 2

[0029] Example 2: Connection of β-glucosidase (bgl1) to cloning vector Taox+PgHT+PB

[0030] 1. The bgl1 target gene and the cloning vector Tao x +PgHT+PB were digested with the restriction enzymes EcoRI and SpeI / XbaI at 37°C for 10 min respectively. The digestion conditions were as follows:

[0031]

[0032] 2. After 1% agarose gel electrophoresis, the digested product was recovered and the two target fragments were respectively recovered and connected with T4 DNA ligase. The ligation system was as follows:

[0033]

[0034]

[0035] Ligase with ligase at 16°C for 12h, the ligation product was transformed into DH5a competent cells and amplified, and the plasmid was extracted with a plasmid extraction kit. After double digestion with EcoRI and PstI, the electrophoresis results showed that there were two bands of 7.0kb and 3.8kb. It indicated that the connection was successful, and it was determined to be a β-glucosidase gene by DNA sequencing.

Embodiment 3

[0036] Example 3: Gene fragment Paox+SS1 is connected to 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 study, and was obtained by double digestion with EcoRI and SpeI endonucleases, purification and recovery;

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

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Abstract

The invention discloses a β-glucosidase with improved trypsin resistance 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. The mutant β-glucosidase (mBGL1) acting on cellobiose and short-chain cellooligosaccharides described in the present invention has resistance to trypsin more than 2 times higher than that of wild-type BGL1, half-life extension, and other enzymatic properties Basically identical to the wild-type enzyme.

Description

technical field [0001] The present invention relates to beta-glucosidases, in particular beta-glucosidases with increased resistance to trypsin. 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 cello-oligosaccharides to generate glucose, remove cellobiose and fiber The feedback inhibition of oligosaccharides on endoglucanase and cellobiohydrolase can also enzymatically hydrolyze the aroma precursors in fruits and tea leaves to enhance aroma. β-Glucosidase belongs to the class of hydrolases and is one of the important components of the cellulase system. [0003] β-Glucosidase has important physiological functions in glycogen degradation in humans and carbohydrate metabolism in animals, plants and microorganisms. In recent years, it has shown important application prospects in the food industry: β-glucosidase can hydrolyze flavor precursor substances in ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56A23L29/00A23K20/189
CPCA23K20/189A23L29/06C12N9/2445C12Y302/01021
Inventor 姚冬生林香娜汪浩谢春芳刘大岭
Owner GUANGDONG GENUIZYMES ANIMAL HEALTH CO LTD
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