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
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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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