A kind of recombinant bacteria and its application in producing cellulase

A cellulase and recombinant bacteria technology, applied in application, recombinant DNA technology, microorganism-based methods, etc., can solve the problems of viscosity of filamentous fungus fermentation broth, reduced cellulase production, high cost, and alleviate the energy crisis. And the effect of environmental pollution, filter paper enzyme activity improvement, and yield improvement

Active Publication Date: 2020-04-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

An important bottleneck that affects the efficiency of submerged fermentation of filamentous fungi is that the fermentation broth of filamentous fungi is very viscous, and local oxygen supply is insufficient, and the synthesis of cellulase from Trichoderma reesei is an energy-consuming process that requires a lot of oxygen. Oxygen deficiency will directly lead to a decrease in cellulase production
Traditional solutions such as increasing the stirring speed or increasing the ventilation volume consume a lot of energy and cost 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 producing cellulase
  • A kind of recombinant bacteria and its application in producing cellulase
  • A kind of recombinant bacteria and its application in producing cellulase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1, the acquisition of recombinant bacteria

[0089] 1. Construction of recombinant expression vector pNOM102-VHb

[0090] 1. Artificially synthesize a vhb gene fragment containing 6 his-tag tags, as shown in sequence 1 of the sequence table, the gene shown in sequence 1 encodes the protein shown in sequence 2.

[0091] In sequence 2 of the sequence listing, amino acid residues 1-146 from the N-terminal are VHB segments, and amino acid residues 147-152 are histidine tags.

[0092] In sequence 1 of the sequence listing, nucleotides 1-438 from the 5' end are the coding gene of the VEGF segment, and nucleotides 439-456 are the coding sequence of the histidine tag.

[0093] 2. Using the pNOM102 plasmid as a template, the primer trpC-F and the primer gpd-R were used for PCR amplification to obtain an expression vector of about 5728 bp containing the gpdA promoter and the TrpC terminator.

[0094] 3. Using a recombination cloning kit to recombine the vhb gene frag...

Embodiment 2

[0125] Embodiment 2, the fermentation application of recombinant bacterium

[0126] Strains to be tested: Trichoderma reesei Tu6, recombinant strain Tu6-vgb + and control recombinant bacteria.

[0127] 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).

[0128] 2. Inoculate 4mL of the spore suspension obtained in step 1 into 400mL of MM liquid medium containing 2% (mass percentage) glucose, cultivate at 200rpm and 28°C, and in the 24h, 36h, 40h, 44h, 48h, At 52h, 56h and 66h, a biosensor analyzer was used to detect the glucose content in the culture medium.

[0129] The result is as Image 6 shown. Image 6 In , the ordinate is the glucose content (mg) per mL of culture medium. The results showed that the recombinant bacteria Tu6-vgb in the medium containing glucose + The rate of sugar consumption was obviously faster than that of Trichoderma reesei Tu6. There was no signif...

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Abstract

The invention discloses recombinant bacteria and applications of the recombinant bacteria in production of cellulase. According to the present invention, the recombinant bacteria are obtained by introducing a specific DNA molecule into Trichodermareesei, wherein the specific DNA molecule has a gpdA promoter and the encoding gene of Vitreoscilla hemoglobin, and the expression of the encoding gene of the Vitreoscilla hemoglobin is initiated by the gpdA promoter; the cellulase yield of the recombinant bacteria is high, and compared to the starting strain, the filter paper enzyme activity is increased by 54.87%, and the extracellular total protein yield is significantly improved; and the great value is provided for the production of cellulase, and the important significance is provided for therelieving of energy crisis and environmental pollution.

Description

technical field [0001] The invention relates to a recombinant bacterium and its application in producing cellulase. Background technique [0002] With the increasing depletion of fossil energy, it is particularly important to develop sustainable alternatives to fossil energy. Lignocellulosic biomass is the most abundant renewable resource on the earth. Cellulase plays a key role in the conversion of lignocellulosic resources. Its yield and activity are directly related to the development and application of new bioenergy. It is of great strategic significance to use cellulase to degrade lignocellulosic biomass into oligosaccharides and monosaccharides, and then efficiently convert them into liquid fuels, which are substitutes for fossil energy. [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 T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/80C12N1/15C12N15/31C12R1/885
CPCC07K14/805C12N9/2437C12N15/80C12N2830/34
Inventor 董志扬林洁陈秀珍张吓妹黄振邦秦丽娜
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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