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A kind of recombinant bacteria and its application in improving the production of cellulase

A technology of cellulase and recombinant bacteria, applied in the direction of application, enzymes, enzymes, etc., can solve problems such as death, viscous fermentation broth, and high cost

Active Publication Date: 2021-06-15
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During submerged fermentation, the fermentation broth is very viscous, which can easily lead to insufficient local oxygen supply, slowing down cell growth or even death, and at the same time, the production of cellulase is significantly reduced
The traditional solution is to increase the stirring speed or increase the ventilation volume, which consumes a lot of energy, costs high and cannot effectively solve the problem of insufficient oxygen supply

Method used

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  • A kind of recombinant bacteria and its application in improving the production of cellulase
  • A kind of recombinant bacteria and its application in improving the production of cellulase
  • A kind of recombinant bacteria and its application in improving the production of cellulase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the acquisition of recombinant bacteria

[0083] 1. Construction of recombinant expression vector pNOM102-TrHb1

[0084] 1. Using the genomic DNA of Trichoderma reesei TU-6 as a template, a primer pair composed of primer Trhb1-F and primer Trhb1-R was used for PCR amplification to obtain a PCR amplification product (Trhb1 gene fragment).

[0085] 2. Using the pNOM102 plasmid as a template, a primer pair consisting of primer trpC-F and primer gpd-R was used for PCR amplification to obtain an expression vector of about 5728 bp containing gpdA promoter and TrpC terminator.

[0086] 3. Using a recombination cloning kit to recombine the PCR amplification product (Trhb1 gene fragment) obtained in step 1 and the expression vector obtained in step 2 to obtain the recombinant expression vector pNOM102-TrHb1 (sequencing verification).

[0087] 2. Trichoderma reesei protoplast preparation

[0088] 1. Trichoderma reesei TU-6 was inoculated into the spore-forming med...

Embodiment 2

[0117] Embodiment 2, the fermentation application of recombinant bacterium

[0118] Strains to be tested: Trichoderma reesei TU-6, recombinant strain TrHb1 and control recombinant strain.

[0119] 1. Suspend the spores of the bacterial strain to be tested with deionized water to obtain a spore suspension (concentration is 10 7 individual / mL).

[0120] 2. Inoculate 4 mL of the spore suspension obtained in step 1 into 400 mL of MM liquid medium containing 2% (mass percentage) glucose, culture at 200 rpm and 28° C. for 48 h, and centrifuge the fermentation broth to collect mycelia.

[0121] 3. Rinse the mycelium obtained in step 2 with sterile water and inoculate it into 100 mL of fermentation medium containing 1% (mass percentage) crystalline cellulose Avicel, culture at 28°C and 200 rpm, and inoculate at 48h, 72h, 96h, 120h and 144h are measured as follows:

[0122] (1) The fermentation system was centrifuged to take the supernatant, and the protein concentration (extracellu...

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Abstract

The invention discloses a recombinant bacterium and its application in improving the production of cellulase. The recombinant bacterium protected by the invention is obtained by introducing specific DNA molecules into Trichoderma reesei. The specific DNA molecule has the coding gene of Trichoderma reesei hemoglobin domain. The present invention identifies the Trichoderma reesei hemoglobin domain for the first time, and successfully expresses the hemoglobin domain in the Trichoderma reesei strain for the first time, improves the fermentation efficiency of the strain, and significantly improves the total extracellular protein production and main cellulase activity . The invention has great value for the production of cellulase, and is of great significance for reducing cost and improving output.

Description

technical field [0001] The invention relates to a recombinant bacterium and its application in improving the production of cellulase. Background technique [0002] Lignocellulosic biomass is the most abundant renewable resource on earth, and its main polysaccharide components—cellulose and hemicellulose—can be efficiently degraded into oligosaccharides and monosaccharides by cellulase and hemicellulase , and then be used in the production of food, feed, bio-energy and other industries. Cellulase plays a key role in this process, and its yield and activity are directly related to the cost and application of food, feed, bioenergy and other related products. [0003] The filamentous fungus Trichoderma reesei is the main industrial strain producing cellulase, which has a strong ability to secrete cellulase and hemicellulase. More than 90% of the world's cellulase products are produced by Trichoderma reesei . At present, the industry mainly uses the submerged fermentation tech...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/31C12N9/42C12R1/885
CPCC07K14/37C12N9/2437
Inventor 董志扬林洁陈秀珍宋冰冉张吓妹
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI