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Method for improving enzyme activity of cellulose of trichoderma reesei

A technology of Trichoderma reesei and enzyme activity, applied in the field of genetic engineering, can solve the problems of limiting the effective utilization of cellulose and low activity

Active Publication Date: 2016-01-27
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

[0006] The extracellular β-glucosidase of Trichoderma reesei accounts for only 1% of its cellulase system, and its activity is relatively low, which also limits the effective utilization of cellulose

Method used

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  • Method for improving enzyme activity of cellulose of trichoderma reesei
  • Method for improving enzyme activity of cellulose of trichoderma reesei
  • Method for improving enzyme activity of cellulose of trichoderma reesei

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of recombinant plasmid pRS424-cbh1-NfBgl3A expressing N.fischeriP1 derived β-glucosidase

[0025] The pRS424-cbh1-NfBgl3A was constructed and transformed into Trichoderma reesei. Under the drive of cbh1 promoter, mRNA was transcribed and translated into CBH1-NfBgl3A fusion protein. The recognition site RDKR sequence of Kex2 protease is designed between CBH1 and NfBgl3A. Therefore, the fusion protein is recognized and cut by Kex2, thereby finally obtaining free CBH1 and β-glucosidase (NfBgl3A).

[0026] The schematic diagram of the plasmid construction of pRS424-cbh1-NfBgl3A is as follows figure 1 shown.

[0027] Amplification of the cbh1 promoter

[0028] Using the genomic DNA of Trichoderma reesei Tu-6 as a template, the cbh1 promoter was amplified with Trcbh1pF (5'-TGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGATATCTAGAGTTGTGAAGTCGG-3') and Trcbh1pR (5'-GCACAATACGACTCCGGCGCTGGCCGATGCGCAGTCCGCGGTTGACTATT-3') primers.

[0029] The PCR reaction conditi...

Embodiment 2

[0041] Example 2. Transformation of the pRS424-cbh1-NfBgl3A plasmid into Trichoderma reesei

[0042] Preparation of pyr4 gene expression cassette DNA

[0043] The pyr4 gene expression cassette was amplified by PCR from the genomic DNA of Trichoderma reesei QM9414, electrophoresed on 1% agarose gel, and the target band in the PCR product was excised, and the DNA was purified for future use. The primers used were: Ppyr4f: 5'-GACTAGACTGACCCCCCCGGTTGGG-3' and Ppyr4r: 5'-CAACTGCATCCAAACCATCTACC-3'.

[0044] The PCR reaction conditions were: 95°C×5min; 32 cycles of 94°C×30s, 55°C×30s, 72°C×1min; 72°C×10min; storage at 4°C. Perform electrophoresis on 1% agarose gel, cut out the target band in the PCR product, and purify the DNA.

[0045] Transformation of Trichoderma reesei Tu-6

[0046] Trichoderma reesei Tu-6 (ATCCMYA-256) was purchased from American Type Culture Collection, USA. Trichoderma reesei Tu-6 was inoculated on a potato medium (PDA) plate, and cultured statically at...

Embodiment 3

[0047] Example 3. Screening of recombinant transformants

[0048] Transformants were grown and selected on MM-glucose agar medium containing 1M sorbitol. A single clone was picked, inoculated in 25ml liquid MM-glucose, and cultured with shaking at 30°C and 180rpm for 1d. Genomic DNA was extracted, and the plasmid pRS424-cbh1-NfBgl3A was successfully transformed into Trichoderma reesei cells by PCR. The primers used in PCR were: Yanzh-Trchb1F (5'-GTTCGGACCCATTGGCAGCACCGG-3') and Yanzh-Nf739R (5'-AAGCGGATACCTAGCGGCGAGTCCTG-3').

[0049] The PCR reaction conditions were: 95°C×5min; 32 cycles of 94°C×30s, 55°C×30s, 72°C×1min; 72°C×10min; storage at 4°C. On 1% agarose gel electrophoresis, there were 7 transformants with DNA bands of the same size as expected (such as figure 2 shown). Therefore, these transformants were positive transformants with pRS424-cbh1-NfBgl3A plasmid. These transformants were inoculated on PDA medium, cultured statically at 30°C for 7 days to produce s...

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Abstract

The invention relates to the field of genetic engineering, in particular to a method for improving the enzyme activity of cellulose of trichoderma reesei. The method includes a step of expressing exogenous beta-glucosidase genes NfBgl3A in the trichoderma reesei. The method has the advantages that heterogenous beta-glucosidase is expressed in the trichoderma reesei, so that transformants for obviously improving the enzyme activity of the cellulose can be obtained; the enzyme activity of filter paper with the cellulose reaches 0.18 U / ml after the transformants are cultivated in MM-Avicel for 6 days and is improved by 3.7 times as compared with original strains; the enzyme activity of CMC (carboxy methylated cellulose) reaches 0.40 U / ml and is improved by 2.9 times as compared with the original strains.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for improving the enzyme activity of Trichoderma reesei cellulase. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose and lignin. Among them, cellulose, as the main component of cell wall construction, is a natural linear macromolecular polymer formed by connecting 8,000-10,000 glucose through β-1,4-glucosidic bonds, with cellobiose as its basic unit. According to statistics, there are currently at least 7×10 11 T Cellulose polysaccharides are produced and are present in large quantities in plant cell walls. Plant cell walls are therefore the most abundant renewable resource on earth and ideal raw materials for the production of renewable biofuels. However, the degradation of cellulosic polysaccharides in plant cell walls into fermentable simple oligosaccharides and monosaccharides such as glucose requires the synergy of c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12R1/885
Inventor 姚斌苏小运薛鲜丽罗会颖王亚茹黄火清柏映国石鹏君王苑
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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