Endoglucanase, its coding gene cel5a-h55 and its application

A cel5a-h55, endoglucanase technology, applied in the field of genetic engineering, can solve problems such as slow progress, achieve the effects of short production cycle, high activity, and ensure product specificity

Inactive Publication Date: 2019-06-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this progress has been rather slow due to a lack of adequate understanding of complex enzyme systems and enzyme-producing bacteria

Method used

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  • Endoglucanase, its coding gene cel5a-h55 and its application
  • Endoglucanase, its coding gene cel5a-h55 and its application
  • Endoglucanase, its coding gene cel5a-h55 and its application

Examples

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Embodiment

[0036] 1. Extraction of total RNA from rumen contents of Hu sheep

[0037] Total RNA was extracted from the rumen contents frozen at -80°C (the following lysis buffer RL, adsorption column RA, protein removal solution RE, rinse solution RW, RNase Free Water were purchased from Adlai Biotechnology Co., Ltd.) in liquid nitrogen. Quickly grind into powder, add 1 ml of lysis solution RL per 50-100 mg of sample, homogenize, and shake vigorously to lyse cells. Incubate at 15-30°C for 5 minutes to completely dissociate ribosomes. Centrifuge at 12,000 rpm for 10 min at 4°C to remove insoluble matter. Transfer the supernatant to a clean centrifuge tube, add 0.2 ml of chloroform to each 1 ml of lysis buffer RL, shake vigorously for 15 s, centrifuge at 12000 rpm for 10 min at 4°C. Transfer the upper colorless aqueous phase to a new tube, add anhydrous ethanol with half the volume of the aqueous phase, mix well, add it to the adsorption column RA, centrifuge at 12,000 rpm for 45s at roo...

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Abstract

The invention belongs to the field of genetic engineering and particularly relates to an endoglucanase and its coding gene cel5A-h55 and application. The gene cel5A-h55 for encoding the endoglucanasehas a nucleotide sequence shown in the formula of SEQ ID No. 1. The bioinformatic analysis result shows that the endoglucanase coded by the gene belongs to the 5th family of glycoside hydrolase. Primers are designed and cloned and then are successfully expressed in a prokaryotic expression system. The recombinant endoglucanase obtained by induced ultrasonic purification has the optimal pH of 5.0 and the optimal temperature of 50 DEG C, keeps the stable enzyme activity at 50 DEG C or less, has strong pH tolerance at pH of 5.0-9.0 and keeps the high enzyme activity. The recombinant endoglucanasehas the large research and industrial application potential.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an endoglucanase, its encoding gene cel5A-h55 and applications thereof. Background technique [0002] Cellulose, as the main component of plant cell wall, is currently the most abundant renewable resource in nature, but its stubborn structure limits its utilization. The process of cellulose degradation into glucose requires endoglucanase (Endoglucanase, EC3.2.1.4), exoglucanase (exoglucanase, E.C.3.2.1.91) and β-glucosidase (β-glucosidase, E.C. 3.2.1.21) The three cellulase systems work together. Among them, endoglucanase is the most important component of the cellulase system. It mainly acts on the amorphous region of cellulose, randomly hydrolyzes the β-1,4 glycosidic bonds in the cellulose chain, and cuts off the long chain of cellulose. It is converted into a large number of short chains with different degrees of polymerization, which reduces the degree of polymerization ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/11C12N15/70C12N1/21C12P19/14C12R1/19
CPCC12N9/2437C12P19/14C12Y302/01004
Inventor 王佳堃何波金舒文高歌李嘉秋
Owner ZHEJIANG UNIV
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