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Endoglucanase, its encoding gene cel5a-h28 and its application

An endoglucanase and gene technology, applied in the field of genetic engineering, can solve problems such as slow process, achieve short production cycle, ensure product specificity, and clear genetic background

Active Publication Date: 2019-10-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 encoding gene cel5a-h28 and its application
  • Endoglucanase, its encoding gene cel5a-h28 and its application
  • Endoglucanase, its encoding gene cel5a-h28 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 rumen content samples frozen at -80°C (the following lysate RL, adsorption column RA, protein-removing solution RE, rinse solution RW, and RNase Free Water were all purchased from Adelaide Biotechnology Co., Ltd.) in liquid nitrogen Grind quickly into powder, add 1ml of lysate RL for every 50-100mg sample, homogenate, shake and mix vigorously to lyse the cells. Incubate at 15-30°C for 5 minutes to completely disassemble the ribosomes. Centrifuge at 12000rpm at 4°C for 10min to remove insoluble matter. Transfer the supernatant to a clean centrifuge tube, add 0.2ml chloroform for every 1ml lysate RL, shake vigorously for 15s, centrifuge at 12000rpm, 4°C for 10min. Take the upper colorless water phase and transfer it to a new tube, add absolute ethanol half the volume of the water phase, mix well and add to the adsorption column RA, centrifuge at 12000rpm for 45s at room temp...

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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-h28 and application. The gene cel5A-h28 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 6.0 and the optimal temperature of 55 DEG C, keeps the stable enzyme activity at 40 DEG C, has strong pH tolerance and keeps the enzyme activity of 85% or more at pH of 4.0-9.0. The recombinant endoglucanase has the large research and industrial application potential.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to an endoglucanase, its coding gene cel5A-h28 and application thereof. Background technique [0002] As the main component of plant cell walls, cellulose is currently the most abundant renewable resource in nature, but its stubborn structure limits its utilization. The process of degrading cellulose 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 act together. Among them, endoglucanase is the most important component of the cellulase system, it mainly acts on the non-crystalline region of cellulose, randomly hydrolyzes the β-1,4 glycosidic bonds in the cellulose chain, cuts off the long chain of cellulose, It is converted into a large number of short chains with different degrees of polymerization, reducing the degree of polymerizatio...

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
Inventor 王佳堃何波金舒文高歌李嘉秋
Owner ZHEJIANG UNIV
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