Heat-resisting acidic cellulase of fungus source and gene and application thereof

A cellulase and gene technology, applied in the field of genetic engineering, can solve the problems of low expression, inappropriate pH range, poor thermal stability, etc.

Active Publication Date: 2015-11-04
INST OF ANIMAL SCI CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although many cellulase enzymes have been cloned and isolated and their properties determined at home and abroad, there are some defects in the properties and characteristics of these enzymes, for example, the pH range is not suitable, the thermal stability is poor, and the expression level is low, which cannot meet the practical application. needs

Method used

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  • Heat-resisting acidic cellulase of fungus source and gene and application thereof
  • Heat-resisting acidic cellulase of fungus source and gene and application thereof
  • Heat-resisting acidic cellulase of fungus source and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Cloning of embodiment 1 cellulase coding gene cel5

[0054] Extraction of Bispora sp.MEY-1 Genomic DNA

[0055] Centrifuge the bacteria cultured in liquid for 3 days at 12,000rpm for 10min, collect the mycelia and extract DNA, wash twice with 70% ethanol, dry in vacuum, add appropriate amount of dd H 2 O was dissolved and stored at -20°C for later use.

[0056] Degenerate primers were designed, and PCR amplification was performed using the total DNA of Bispora sp.MEY-1 as a template. A fragment of about 451bp was obtained, which was recovered and sequenced.

[0057] TAIL-PCR primers uspl, usp2, us3; dsp1, dsp2, dsp3 were designed according to the nucleotide sequence obtained by sequencing (see Table 1). The flanking sequence of the known gene sequence was obtained by TAIL-PCR, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing. The correctly sequenced fragments were spliced ​​to obtain the full-length gene.

[0058] Tab...

Embodiment 2

[0063] The construction of embodiment 2 cellulase engineering strains

[0064] (1) Construction of expression vector and expression in yeast

[0065] Using the cDNA of the correctly sequenced cellulase Cel5 as a template, primers F and R (see Table 1) with EcoR I and Not I restriction sites were designed and synthesized to amplify the coding region of the mature protein of Cel5. increase. And utilize EcoR I and Not I to cut PCR product, link into expression vector pPIC9 (Invitrogen, San Diego), the sequence of cellulase Cel5 mature protein is inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, and signal peptide forms correct The reading frame was constructed into a yeast expression vector pPIC9-cel5, and transformed into Escherichia coli competent cell Trans1. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmids....

Embodiment 3

[0069] The preparation of embodiment 3 recombinant cellulase

[0070] (1) Massive expression of cellulase gene Cel5 at shake flask level in Pichia pastoris

[0071] The transformant with high enzyme activity was screened out, inoculated into a 1L Erlenmeyer flask with 300mL of BMGY liquid medium, cultured on a shaking table at 30°C at 220rpm for 48h; centrifuged at 5,000rpm for 5min, discarded the supernatant gently, and then added 100mL containing 0.5% methanol BMMY liquid medium, 30°C, 220rpm induction culture for 72h. During the induction culture period, add methanol solution once every 24 hours to compensate for the loss of methanol, and keep the methanol concentration at about 0.5%; (3) Centrifuge at 12,000×g for 10 minutes, collect the supernatant fermentation liquid, detect the enzyme activity and perform SDS-PAGE protein Electrophoretic analysis.

[0072] (2) Purification of recombinant cellulase

[0073] The supernatant of the recombinant cellulase expressed in the...

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Abstract

The invention relates to the field of gene engineering, in particular to heat-resisting acidic cellulase of a fungus source and a gene and application thereof. The amino acid sequence of the heat-resisting acidic cellulase is showed as in SEQ ID NO.1 or SEQ ID NO.2. According to the novel gene of the cellulase, the coded cellulase of the novel gene has good performance and can be applied to industries of feedstuff, foods, medicine and others. According to the technical scheme, by the means of gene engineering, the cellulase excellent in performance and suitable for industrial application can be produced.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to a heat-resistant acid cellulase derived from fungi and its gene and application. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Cellulose is an important polysaccharide. It is the material of plant cell support material and the most abundant biomass resource in nature. The structure of cellulose is determined as β-D-glucose units connected by β-(1→4) glycosidic bonds. The resulting linear polymer has no branches in the structure, which can be degraded to glucose by cellulase. [0003] Cellulase refers to the general term for a group of enzymes that can hydrolyze glucosidic bonds and decompose cellulose into cellobiose and glucose. The hydrolysis process of cellulose mainly includes three steps: the first step is that the endo-cellulase acts on the amorphous region inside the cellulose,...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2437
Inventor 姚斌罗会颖郑菲王苑王亚茹黄火清石鹏君柏映国苏小运孟昆马锐
Owner INST OF ANIMAL SCI CAAS
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