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Cellulose Cel5A with wide pH range as well as gene and application

A technology of cellulase, ppic9-cel5a, applied in the field of genetic engineering, can solve problems such as not being able to meet industrial requirements

Active Publication Date: 2013-05-01
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the optimum pH of cellulase produced by Trichoderma, which is widely used in industry, is about 5.0, and the optimum temperature is between 50-60°C, which cannot meet the industrial requirements of cotton fabric washing and finishing industry, paper making and pulping industry.

Method used

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  • Cellulose Cel5A with wide pH range as well as gene and application
  • Cellulose Cel5A with wide pH range as well as gene and application
  • Cellulose Cel5A with wide pH range as well as gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] The enzyme production property test of embodiment 1 fungus Bispora antennata CBS 126.38

[0121] After Bispora antennata CBS 126.38 was cultured in malt extract medium, it was inoculated in induction medium (0.2% MgSO 4 ·7H 2 O, 0.1% KH 2 PO 4 , 0.1% CuSO 4 ·5H 2 O, 0.1% CaCl 2 , 0.5% peptone, 1% corncob flour, 1% bran, pH 5.0), cultivated at 30°C for 3-4 days, and measured the activity of cellulase and hemicellulase. The bacteria was determined to be a high cellulase producing strain.

Embodiment 2

[0122] Example 2 Cloning of the fungal Bispora antennata CBS 126.38 cellulase-encoding gene cel5A

[0123] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0124] According to the conserved sequence of the glycoside hydrolase gene (YEM / FHQYLD and WWAG / AGPW), the degenerate primers P1 (5'-...

Embodiment 3

[0134] Obtaining of the cDNA sequence of embodiment 3 cellulase gene

[0135] The total RNA of Bispora antennata CBS 126.38 was extracted, a strand of cDNA was obtained by reverse transcriptase, and then the appropriate primers Cel5AF (5'-ATGCGCTTTCCAACCCCCCTCCTCGC-3') and Cel5AR (5'-CTACACATACTTTGTTAAAATATCAATGTATCCCTCATAAGCCC-3') were designed to amplify the single strand Strand cDNA to obtain the cDNA sequence of cellulase, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0136] After comparing the genome sequence and cDNA sequence of cellulase, it was found that the gene of cellulase was analyzed by the genome DNA sequence and cDNA sequence of cellulase. They are respectively: 592-640bp and 974-1,078bp, and the cDNA length is 1041bp. The N-terminal 18 amino acids are its predicted signal peptide sequence. The cellulase Cel5A cDNA sequence encoded protein was homologously compared with the sequence on GeneBank, and it was...

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Abstract

The invention relates to the field of genetic engineering, in particular to a cellulose Cel5A with wide pH range as well as a gene and an application. The invention provides a cellulose Cel5A with wide pH range, wherein the cellulose Cel5A has an amino acid sequence shown by SEQ ID NO.1 or 2. And the invention also provides a gene for encoding the cellulose Cel5A with wide pH range, as well as a recombinant vector, a recombinant strain and an application thereof, wherein the nucleotide sequence of the gene is shown by SEQ ID NO.3, 4 or 5, and the recombinant vector and the recombinant strain both contain the genes. The cellulose Cel5A with wide pH range, provided by the invention, has the advantages of extremely good thermal resistance, can be applied to feed industry, food industry, medicine industry and the like for animals and fishes, and can be also applied to papermaking and textile and printing industries.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a wide pH range cellulase Cel5A and its gene and application. Background technique [0002] Cellulose is a polysaccharide composed of glucose connected by β-1,4 glycosidic bonds, and is one of the most important renewable resources in nature. The utilization of these renewable resources is an important part of the degradation of cellulose. The degradation of cellulose requires the synergy of three enzymes, including: endo-1 cellulase (endo-1, 4-β-d-glucanase, EC 3.2.1.4, referred to as EG), which preferentially acts on the interior of cellulose molecules The non-crystalline region of β-1,4-glucosidic bonds is randomly hydrolyzed, the long-chain cellulose molecules are truncated, and a large number of small molecule celluloses with non-reducing ends are produced; exo-cellulase (exo-1,4- β-d-glucanase or cellobiohydrolase, EC 3.2.1.91, referred to as CBH)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N15/81C12N1/21C12N1/15C12N1/19C12R1/84C12R1/19C12R1/645
Inventor 姚斌杨培龙罗会颖刘琼王亚茹黄火清石鹏君柏映国孟昆袁铁铮
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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