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Fungus cellulose enzyme system constitution/characteristic regulation gene and application thereof

A technology for regulating genes and cellulase, which is applied in the field of genetic engineering and can solve problems such as low enzyme activity

Inactive Publication Date: 2013-02-13
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in biomass conversion, the cellulase liquid β-glucosidase secreted by fungi has low enzyme activity, which usually leads to the accumulation of cellobiose, causing its feedback inhibition of the enzyme activity of cellobiohydrolase and endonuclease

Method used

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  • Fungus cellulose enzyme system constitution/characteristic regulation gene and application thereof
  • Fungus cellulose enzyme system constitution/characteristic regulation gene and application thereof
  • Fungus cellulose enzyme system constitution/characteristic regulation gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Obtaining of fungal cellulase enzyme activity regulation gene knockout cassette:

[0054] (1) Cultivate the Penicillium decumbens strain on the bran medium for three days, then wash off the spores with normal saline, and follow the 1×10 8 Proportionally inserted into the basic medium, 200rpm, 30°C for two days, centrifuged at 4000rpm, collected bacteria, collected in a 1.5ml centrifuge tube;

[0055] (2) Add 500 μl of extraction buffer and 0.1 g of quartz sand to the centrifuge tube, vortex vigorously for 1 min to disperse the bacteria in the extraction buffer, place at 65°C for 20 min; add 500 μl of phenol / chloroform (mixing ratio 1 1) Vortex vigorously for 30s, centrifuge at 12000rpm for 10min at room temperature, and collect the supernatant;

[0056] Transfer the supernatant to another sterile 1.5ml centrifuge tube, add 0.1 times the volume of 3M NaAc solution (pH 4.8) and 0.6 times the volume of isopropanol, mix it upside down, and place it at -20°C for 15 minutes;...

Embodiment 2

[0107] Obtaining of fungal cellulase enzyme activity regulation gene knockout cassette:

[0108] (1) Cultivate the Trichoderma reesei QM9414 strain on the bran medium for three days, then wash off the spores with physiological saline, and follow the 1×10 8 Proportionally inserted into the basic medium, 200rpm, 30°C for two days, centrifuged at 4000rpm, collected bacteria, collected in a 1.5ml centrifuge tube;

[0109] (2) Add 500 μl of extraction buffer and 0.1 g of quartz sand to the centrifuge tube, vortex vigorously for 1 min to disperse the bacteria in the extraction buffer, place at 65°C for 20 min; add 500 μl of phenol / chloroform (mixing ratio 1 1) Vortex vigorously for 30s, centrifuge at 12000rpm for 10min at room temperature, and collect the supernatant;

[0110] Transfer the supernatant to another sterile 1.5ml centrifuge tube, add 0.1 times the volume of 3M NaAc solution (pH 4.8) and 0.6 times the volume of isopropanol, mix it upside down, and place it at -20°C for ...

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PUM

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Abstract

The invention relates to a fungus cellulose enzyme system constitution / characteristic regulation gene and application thereof. Nucleotide sequences are demonstrated in SEQ ID No.1 or SEQ ID No.4. The invention further relates to fungus cellulose enzyme activity regulation factors coded by fungus cellulose enzyme activity regulation genes, and the application of the fungus cellulose enzyme activity regulation genes in preparation of special fungus cellulose enzyme. Amino acid sequences are demonstrated in SEQ ID No.2 or SEQ ID No.5. The fungus cellulose enzyme activity regulation genes and the fungus cellulose enzyme activity regulation factors can be used for regulating enzyme activity of cellulose enzyme in fungus. After a knockout box replaces the fungus cellulose enzyme activity regulation genes, compared with original strains, the enzyme activity of cellulose enzyme can be changed about 20% so as to meet requirements of various industries for the enzyme activity of different cellulose enzyme.

Description

technical field [0001] The invention relates to a fungal cellulase enzyme system composition / characteristic regulation gene and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] The cellulases secreted by fungi mainly include exoglucosidase, endoglucosidase and β-glucosidase. Cellulase solution can be used in biomass conversion, textile and food industries. Different application directions have different requirements for the proportion of enzymes in the enzyme solution. [0003] In certain aspects, higher β-glucosidase enzyme activity is desired. For example, in biomass conversion, the cellulase liquid β-glucosidase secreted by fungi has low enzymatic activity, which usually leads to the accumulation of cellobiose, causing its feedback inhibition of the enzymatic activities of cellobiohydrolase and endonuclease. Therefore, the proportion of β-glucosidase in the enzyme liquid can be increased, the accumulation of ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N9/42C07K14/385C07K14/37
Inventor 方诩王方忠王明钰侯少莉刘奎美韩丽娟
Owner SHANDONG UNIV
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