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Acidic cellulose from fungus and gene and application thereof

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

Active Publication Date: 2015-12-16
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, 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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  • Acidic cellulose from fungus and gene and application thereof
  • Acidic cellulose from fungus and gene and application thereof
  • Acidic cellulose from fungus 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 Prostheciumopalus Genomic DNA

[0055] The bacteria cultured in liquid for 3 days were centrifuged at 12,000rpm for 10min, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new, packed Put 15ml of CTAB lysate in a 50mL centrifuge tube, gently mix it up and down, place it in a water bath at 65°C for 3 hours, and mix it upside down gently every 20 minutes to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the supernatant and add an equal volume of phenol / chloroform for extraction, and place it at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 10 ...

Embodiment 2

[0060] The acquisition of embodiment 2 cellulase cDNA

[0061] Extract Prostheciumopalus total RNA using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then design primers F and R (see Table 1) for amplifying the open reading frame, amplify the single-stranded cDNA, obtain the cDNA sequence of cellulase, and amplify to obtain product recovery Sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0062] After comparing the genome sequence and cDNA sequence of cellulase, it was found that the gene contained an intron, the cDNA was 993 bp long, encoded 330 amino acids and a stop codon, and the N-terminal 19 amino acids were its signal peptide sequence. The comparison proves that the cellulase-encoding gene isolated and cloned from Prosthecium opalus is a new gene.

Embodiment 3

[0063] The construction of embodiment 3 cellulase engineering strains

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

[0065] Using the correctly sequenced cellulase Cel5 cDNA as a template, primers F and R (see Table 1) with EcoRI and NotI restriction sites were designed and synthesized to amplify the coding region of the mature protein of Cel5. And use EcoRI and NotI to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, SanDiego), the sequence of the cellulase Cel5 mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and form a correct reading frame with the signal peptide, The yeast expression vector pPIC9-cel5 was constructed 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. Lineari...

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Abstract

The invention relates to the field of genetic engineering, in particular to acidic cellulose from a fungus and a gene and application thereof. The amino acid sequence of the acidic cellulose is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention further provides the novel acidic cellulose gene. The acidic cellulose encoded by the acidic cellulose gene has good property and can be applied to feeds, foods, pharmaceuticals and other industries. By means of the technical scheme, the acidic cellulose having excellent property and suitable for industrial application can be produced by adopting a genetic engineering means.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to an acid cellulase derived from a fungus 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, and then h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2437
Inventor 姚斌罗会颖郑菲苏小运石鹏君柏映国黄火清王亚茹孟昆师霞马锐
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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