Acidic high-temperature-resistant cellulase Cel5 and gene and application thereof

A technology of cellulase and high temperature resistance, applied in the field of genetic engineering, can solve the problems of poor thermal stability, inappropriate pH range, low expression level, etc., and achieve the effect of easy fermentation and production

Active Publication Date: 2017-07-21
INST OF ANIMAL SCI CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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, such as inappropriate pH range, poor thermal stability, and low expression level, which cannot meet practical applications. needs

Method used

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  • Acidic high-temperature-resistant cellulase Cel5 and gene and application thereof
  • Acidic high-temperature-resistant cellulase Cel5 and gene and application thereof
  • Acidic high-temperature-resistant cellulase Cel5 and gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] Cloning of embodiment 1 cellulase coding gene cel5

[0052]Genomic DNA of Talaromyces leycettanus JCM12802 was extracted, and the total DNA of Talaromycesleycettanus was used as a template, and cel-F and cel-R were used as primers for PCR amplification (see Table 1). The PCR reaction parameters were: 95°C for 5min; 94°C for 30sec, 60°C for 30sec , 72°C for 30 sec, 30 cycles, a fragment of about 1400bp was obtained, which was recovered and sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0053] Table 1 Primers required for this experiment

[0054]

Embodiment 2

[0055] The acquisition of embodiment 2 cellulase cDNA

[0056] Extraction of total RNA from Talaromyces leycettanus JCM12802, using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, and then use cel5-F and cel5-R (see Table 1) to amplify the single-stranded cDNA to obtain the cDNA sequence of cellulase. The amplified product is recovered and sent to Ruibo Sequencing Biotech Ltd.

[0057] After comparing the genome sequence and cDNA sequence of cellulase, it was found that the gene contained 4 introns, the cDNA was 1230 bp long, encoded 409 amino acids and a stop codon, and the N-terminal 18 amino acids were its signal peptide sequence. The comparison proves that the cellulase-encoding gene isolated and cloned from Talaromyces leycettanus JCM12802 is a new gene.

Embodiment 3

[0058] The construction of embodiment 3 cellulase engineering strains

[0059] (1) Construction of expression vector and expression in yeast

[0060] With the cDNA of cellulase cel5 correctly sequenced as a template, primers cel5-f and cel5-r (see Table 1) with SnaB I and Not I restriction enzyme sites were designed and synthesized, for the mature protein of Cel5 The coding region was amplified, and the PCR product was digested with SnaB I and Not I, and connected into the expression vector pPIC9 (Invitrogen, San Diego), and the sequence of the cellulase Cel5 mature protein was inserted into the downstream of the signal peptide sequence of the above expression vector, Form the correct reading frame with the signal peptide, construct the yeast expression vector pPIC9-cel5, and transform 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 rec...

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Abstract

The invention relates to the field of genetic engineering, in particular to an acidic high-temperature-resistant cellulase Cel5 and a gene and application thereof. The amino acid sequence of the cellulase is shown as SEQ ID NO.1 or SEQ ID NO.2. The invention provides a new cellulase gene. The cellulase encoded by the gene has good properties, can be applied to feeds, foods, medicines and other industries. According to the technical scheme, it can be achieved that the cellulase having good properties and suitable for industrial application is produced by utilizing a gene engineering means.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to acid high temperature resistant cellulase Cel5 and its gene and application. Background technique [0002] 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 hydrolyzes the β-(1→4) glycosidic bond to shorten the cellulose molecule, Then the exo-cellulase acts on the end of the cellulose linear molecule to hydrolyze the β-(1→4) glycosidic bond, cutting off one cellobiose molecule at a time, and finally, the glucosidase hydrolyzes the cellobiose into glucose molecular. [0003] Cellulase has been widely used in many fields such as food, medicine, feed, papermaking, textile printing and dyeing, petroleum exploration, f...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12R1/84
CPCC12N9/2437C12N15/815
Inventor 姚斌罗会颖郑菲涂涛苏小运黄火清王苑柏映国王亚茹孟昆
Owner INST OF ANIMAL SCI CAAS
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