Acidic cellulase EGI, gene thereof and application thereof

A technology of acid cellulase and cellulose, applied in the field of genetic engineering

Active Publication Date: 2013-03-20
北京挑战生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A variety of cellulase genes have been successfully expressed (Saloheimo et al. Eur. J. Biochem, 1991, 249: 584-591; Liu Yan et al. Feed Industry, 2007, 28 (18): 11-14), but It still cannot fully meet the needs of industrial production in terms of expression amount and properties. For example, cellulase for feed requires an optimal pH value of acidic and at the same time has high activity in the acidic and neutral ranges, and good thermal stability. High activity at room temperature

Method used

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  • Acidic cellulase EGI, gene thereof and application thereof
  • Acidic cellulase EGI, gene thereof and application thereof
  • Acidic cellulase EGI, gene thereof and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0151] The isolation of the genomic DNA of embodiment 2 Penicillium sp.TZ1 bacterial strains

[0152] The mycelium cultured in potato juice medium for 3 days was filtered with sterile filter paper and placed in a mortar, 2 mL of extract was added, ground for 5 min, then the ground liquid was placed in a 50 mL centrifuge tube, and genomic DNA was extracted by CTAB method (Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd Edition 2001)).

[0153] Cloning of Example 3 Penicillium sp.TZ1 Cellulase Encoding Gene

Embodiment 2

[0154] The conserved cellulase sequence of the 7th family of glycoside hydrolases was determined through the Pfam database, ClustalW software and published information about the cellulase of the 7th family of glycoside hydrolases. At the same time, combined with the published information on the sequence characteristics of filamentous fungi, the conserved amino acid sequence was determined as GYCDAQC and GCGFNPY, and then deduce the corresponding nucleotide sequence according to the genetic code of the species.

[0155] The degenerate primers EG7F (5'-GGYTACTGYGAYGCNCARTG-3') and EG7R (5'-TATGGGRTTRAANCCCRCANCC-3', Y=C / T, R=A / G, N=A / T / C / G).

Embodiment 3

[0156] Genomic DNA of Penicillium sp.TZ1 was used as a template, and EG7F and EG7R were used as primers for PCR amplification. Then the PCR product was detected by agarose gel electrophoresis, and the obtained fragment of about 250bp was recovered by the recovery kit and ligated into pEASY-T 3 On the carrier, the ligation product was heat-shocked and transformed into E. coli competent cells, and the recombinant transformants were screened, and the transformants containing the target fragment were sent to Beijing Biomed Biotechnology Company for sequencing.

[0157] According to the sequencing results, BLASTx was used to compare the sequences in GenBank of NCBI, and it was preliminarily determined that the obtained fragments were gene fragments of cellulase of the seventh family of glycoside hydrolases.

[0158] According to the obtained partial sequence of the cellulase, three upstream and three downstream specific primers were respectively designed, and the upstream and downs...

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Abstract

The invention relates to the field of genetic engineering, and specifically relates to an acidic cellulase EGI, a gene thereof and an application thereof. The invention provides a novel acidic cellulase EGI with an amino acid sequence represented by SEQ ID NO.1 or 2. The invention also provides a gene encoding the acidic cellulase EGI. The nucleotide sequence of the gene is represented by SEQ ID NO.3, 4 or 5. The invention further provides a recombinant vector and a recombinant strain containing the gene, and an application thereof. The effective pH value range of the cellulase EGI provided by the invention is wide, wherein the pH value is ranged from 2.0 to 6.0, and an optimal pH value is 2.5 to 4.0. An optimal temperature is 50 DEG C. Under an acidic condition, the cellulase EGI has good stability and heat resistance. The cellulase EGI can be used in various industries such as feedstuff industries and foodstuff industries.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acid cellulase EGI and its gene and application. Background technique [0002] Cellulose is the most abundant renewable biomass resource in nature. It consists of several hundred to tens of thousands of pyranoid D-glucose residues connected by β-1,4-glycosidic bonds to form a cellulose chain. The cellulose chains associate with each other through hydrogen bonds to form cellulose bundles, which are homopolysaccharides insoluble in water (Beguin. J Microbiol, 1990, 44: 219-248). It is of great significance to the sustainable development of human society to convert these huge renewable resources into energy, food and chemical raw materials urgently needed by human beings (Wang Jianping, Journal of Zhejiang Fisheries Institute, 1996, 15(2): 140 ~144). The utilization of these renewable resources, the degradation of cellulase (cellulase) is a very importa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/19C12N1/14A23K1/165A23L1/305C12R1/84C12R1/80A23K20/189A23L33/175
Inventor 刘建平王晓睿谢宁杨禄良黄辉
Owner 北京挑战生物技术有限公司
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