Preparation method for recombination strains for generating Beta glucosaccharase

A technology of glucosidase and construction method, which is applied in the field of construction of recombinant bacteria producing β-glucosidase, which can solve the problems of low enzyme activity, difficult extraction, complex components of β-glucosidase, etc.

Active Publication Date: 2010-02-24
安徽丰原集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the enzyme activity of β-glucosidase in liquid fermentation is generally low; while the composition of β-glucosidase produced by solid-state fermentation is complex and difficult to extract

Method used

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  • Preparation method for recombination strains for generating Beta glucosaccharase
  • Preparation method for recombination strains for generating Beta glucosaccharase

Examples

Experimental program
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Embodiment 1

[0018] 1. Extraction of Aspergillus niger mRNA

[0019] 1. Aspergillus niger total RNA extraction:

[0020] (1) After grinding the mycelium into powder in liquid nitrogen, add 1 mL of Trizol liquid nitrogen per 50-100 mg of mycelium for grinding, and note that the total sample volume cannot exceed 10% of the volume of Trizol used.

[0021] (2) The grinding solution was placed at room temperature for 5 minutes, then chloroform was added at a ratio of 0.2 mL per 1 mL of Trizol solution, the centrifuge tube was tightly capped, and the centrifuge tube was shaken vigorously by hand for 15 seconds.

[0022] (3) Take the upper aqueous phase into a new centrifuge tube, add isopropanol at a ratio of 0.5 mL of isopropanol per mL of Trizol solution, place at room temperature for 10 minutes, and centrifuge at 12,000g for 10 minutes.

[0023] (4) Discard the supernatant, add at least 1 mL of 75% ethanol per 1 mL of Trizol solution, mix by vortexing, and centrifuge at 7500g for 5 minutes a...

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Abstract

The invention discloses a preparation method for recombination strains for generating Beta glucosaccharase, comprising the steps of extracting gross RNA from strains which can provide Beta glucosaccharase gene, acquiring Beta glucosaccharase c DNA sequence by utilizing RT-PCR technique, attaching the c DNA sequence to a carrier to acquire a recombinant plasmid transferred host to form the recombination strains for generating Beta glucosaccharase. By adopting the preparation method of the invention, Pichia pastoris which can highly generate Beta glucosaccharase is prepared and is applied to generating Beta glucosaccharase, with high industry using value.

Description

technical field [0001] The invention relates to a construction method of a recombinant bacterium producing β-glucosidase, in particular to a recombinant Pichia pastoris capable of overexpressing β-glucosidase and its application. Background technique [0002] The English name of β-glucosidase (EC 3.2.1.21) is β-glucosidase, which belongs to the class of hydrolase, also known as β-D-glucoside hydrolase, alias gentiobiase, cellobiase and amygdalin enzymes. It catalyzes the hydrolysis of the terminal non-reducing β-D-glycosidic bond while releasing the ligand and glucosome. β-Glucosidase degrades various oligosaccharides and cellobiose into glucose in the process of cellulase hydrolysis, which is the key enzyme in the last step and plays a key role in the saccharification and hydrolysis of cellulose. β-glucosidase also has a transglycosidase effect, which can synthesize two glucose molecules into one sophorose molecule through transglycosidase under certain conditions. Sopho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/81C12N1/19C12N9/42C12P19/14C12P19/12C12R1/84C12R1/685C12R1/66C12R1/69C12R1/885C12R1/645C12R1/72C12R1/30C12R1/01
Inventor 李荣杰徐斌薛培俭段绪果
Owner 安徽丰原集团有限公司
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