High Activty Cellulase Engineering Strain Construction By The Method Of DNA Recombinantion

Cellulose is the most abundant renewable nature product in the biosphere. Annual production of cellulose is estimated to be 10-50×109 tons. Presently, most of cellulose are wasted and pollute environment. So it has great realistic meaning for human being to solve energy crisis, food shortage and environment pollution that using cellulose produced by microorganism transform cellulose to energy, food and chemical.During this paper, a cellulase-producing strain was screened from ecological forest in Huangshan, on basis of that, taxonomy position, and the construction of cellulase gene had been studied, so as to construct genetically cellulase engineering strain. The major results are as follows:A strain with high activity of cellulase was sparated from rotten wood of ecological forest by the mothed of CMC-congon red plate screening. During the mothed of the mutagenesis on protoplast by Ultraviolet, a high activity of cellulase strain had been obtained and named TP-02; the Filter Paper Activity (FPA) and Endoglucanase (CMCase) of the strain could be achieved 7.56 IU/mL and 28.5IU/mL respectively, the fermentation time advanced 36h compared with the current study strain Trichoderma reesei QM9414.The mutant TP-02 was identified by utilization of the methods of both molecular biological identification and morphological identification. The results of that demonstration that the strain used in this paper, belong to Zygomycotina, Phycomycetes, Mucorales, Mucoraceae, Rhizopus stolonifer var.reflexus.The first stand cDNA synthesised by using the Rhizopus stolonifer var.reflexus mRNA template, and guiding with primer Oligod (T) 18, under the condition of reverse transcriptase. The endo-glucanase gene sequences of the related species were searched to design the primers by softares to amplify the first stand cDNA. The sequence identified as new gene encoding Endo-glucanase by the program of Blast in NCBI, and submitted to GenBanK, and a receiving number of the sequence FJ807269 was obtained. Plasmid pET20b-EG1 was constructed by ligation the PCR products to the prokaryotic expression vector pET20b, and then transfected it into E. coli BL21 (DE3). After 36h fermentation and induction by IPTG, the recombinant bacteria's Endo-glucanase activity could achieve the peak of 0.715IU/mL. a band with molecular weight about 40 kDa could be detected by SDS-PAGE of the purified fermentation product.Under the condition of reverse transcriptase, double-stranded length cDNA was synthesized by utilizing Rhizopus stolonifer var.reflexus TP-02 mRNA template, and guiding with Anchor primer 5'-(GA) 10 CTCGAGCGGC CGC (T) 18 V-3'. Then an adaptor containing EcoR I site was joined with that by the ligation kit. After that, the double-stranded cDNA with both EcoR I and Not I site was digested by restriction endonuculease Not I and linked into double digested vector pPIC9K. After that, the plasimid was transfected into Pichia pastoris GS115 competent cells to construct cDNA library. The positive clones were sceened by the mothed of CMC-congon red plate from the cDNA library. Three different sequences were obtained after double digested and sequenced the positive clones. One sequence showed 100% Similarity with the sequence FJ807269, others were new gene encoding Endo-gluancanase, after alignment analysis, and submitted to GenBank with the accession number:HM043656 and HM043657. After 60h fermentation and induction by methanol, the recombinant Yeasts' Endo-glucanase activity could achieve the peak of 1.15 IU/mL 1.86IU/mL and 1.56IU/mL. Three bands with molecular weight about 40 kDa 44 kDa,46kDa could be detected by SDS-PAGE of the purified fermentation product corresponding to the sequence of FJ807269, HM043656, and HM043657.