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Endoglucanase as well as encoding gene and use thereof

A technology of endoglucanase and coding gene, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of low efficiency, high cost, high price, etc.

Inactive Publication Date: 2008-07-23
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the wide use of cellulase and the need to use cellulase with different properties for different purposes, and because of the low efficiency and high price of cellulase, the cost of producing fuel alcohol from cellulose is too high to be truly To achieve industrialization, therefore, new cellulase enzymes are required

Method used

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  • Endoglucanase as well as encoding gene and use thereof
  • Endoglucanase as well as encoding gene and use thereof
  • Endoglucanase as well as encoding gene and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1, the acquisition of endoglucanase Umcel5D and its coding gene

[0042] 1. Construction of metagenomic library of buffalo rumen uncultured microorganisms

[0043] 1. Extraction of uncultured microbial metagenomics from buffalo rumen

[0044] Get 50g buffalo rumen contents (the source of the sample is the rumen of the buffalo just slaughtered by Nanning Meat Co., Ltd., and the collected sample is immediately preserved with liquid nitrogen), suspended in 200ml of 0.18M potassium phosphate buffer (pH6.5), Gently oscillate evenly, let stand for 10 minutes, let the cellulose particles in the rumen content automatically precipitate, discard the supernatant, and then add 200ml of 0.18M potassium phosphate buffer (pH6.5) to wash the sediment twice, and the last time Metagenomic DNA of microorganisms adsorbed to cellulose pellets was extracted by direct extraction from pellets. Add 100ml of extraction buffer (100mM sodium phosphate pH8.0; 100mM Tirs-HCl pH8.0; 100m...

Embodiment 2

[0063] Embodiment 2, expression of umcel5D in Escherichia coli

[0064] 1. Construction of a recombinant vector (pET-umcel5D) capable of expressing umcel5D

[0065] After the gene with coding signal peptide is expressed in vivo, the expression product is likely to be secreted outside the cell. In order to prevent the expression product from being secreted outside the cell, only the 295th to 1879th base at the 5' end of the artificially synthesized sequence 1 (that is, the DNA sequence in the umcel5D gene except for the coding signal peptide functional domain and the stop codon), 1585bp , it is estimated that the molecular weight of the protein encoded by the synthesized fragment is 59.1202 KDa, and the isoelectric point pI is 5.92.

[0066] Artificially synthesize the entire sequence of the umcel5D gene except for the signal peptide and the stop codon (i.e., the nucleotide sequence at the 295th-1879th position at the 5' end of sequence 1), and the two ends of the synthetic um...

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Abstract

The invention discloses an endoglucanase and a coding gene of the endoglucanase and an application of the endoglucanase and the coding gene. The endoglucanase is one type of the protein with the following amino acid residue sequence: 1) SEQ ID No.: 2 in the sequence table; 2) the amino acid residue sequence of the SEQ ID No.: 2 in the sequence table forms a protein with the endoglucanase activity by the substitution and / or the deletion and / or the addition of one or a plurality of amino acid residues. The endoglucanase and a coding gene of the endoglucanase has the advantages that: the endoglucanase has very high carboxymethyl cellulose enzyme activity (4029U / g), and the enzyme has a wide PH value range and a wide temperature range. The endoglucanase and the coding gene of the endoglucanase can be widely used in the degradation of cellulose.

Description

technical field [0001] The invention relates to an endoglucanase, its coding gene and application. Background technique [0002] Cellulose is mainly the most abundant renewable biomass (biomass) resource on earth synthesized by plants using carbon dioxide and water through photosynthesis under the action of solar energy. According to reports, the global annual production of cellulose through photosynthesis is as high as 1.55×10 9 tons, 89% of which have not been utilized by humans (Dunlap C, Chiang GC. Utilization and recycle of agriculture wastes and residues. Shuler M L. Boca Raton, Florida. USA: CRC Press Inc. 1980.19). Cellulose is a polymer composed of multiple glucose residues connected by β-1,4-glucosidic bonds, and its basic repeating unit is cellobiose. The basic structure of natural cellulose is composed of microfibril bundles composed of fibrils. Fibrils are composed of 15-40 long chains of cellulose molecules with crystalline and non-crystalline regions. The ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
Inventor 冯家勋段承杰赵广存普连仙冼亮庞浩唐纪良
Owner GUANGXI UNIV
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