Ferment Conditions Optimizing For Producing Cellulase And Cloning Of CBHⅠ Of Penicillium Oxalicum Currie & Thom

Cellulase is a class of multi-component enzymes which can degradate cellulose to glucose, including exoglucanase (C1 enzyme), endoglucanase (CX enzymes) andβ-glucosidase. Cellulase has a very broad application prospects in the field of food, brewing industry, deep processing of agricultural and sideline products, fodder, medicine, environmental protection and chemical sectors, as well as the use of renewable energy.Goose origin Penicillium oxalicum Currie & Thom F67 was selected and the liquid ferment cultivation was adopted for the experiment, the CMCase activity, Cotton lyase activity,β-glucosidase activity and FPA activity were tested to explore the optimum composition of substrate (the carbon source and nitrogen source) and ferment condition (inoculum's size, temperature, pH and ferment time) of Producing Cellulase. Furthermore, the optimum ferment condition was screened by the orthogonal design for the different constituents of cellulase. At the same time, the CBHⅠgene of exoglucanase was cloned and the eukaryotic expression vector was constructed by the molecular biology techniques such as RT-PCR, degenerate PCR and TAIL-PCR. Beside, the biological information were analyzed by bioinformatics tools which laid foundation for the genetic characteristics and the Construction of high-performance cellulose decomposing bacteria.The findings indicated that:1. The result of liquid fermentation characteristics research of goose origin Penicillium oxalicum Currie & Thom F67 was showed that: CMC-Na(proper recruitment was 0.9g~1.2g/100mL)could strongly induced the penicillium oxalicum to produce cellulase (P<0.01); When NH4NO3(proper recruitment was 0.6g~1.2g/100mL)was used as nitrogen source, both of enmyze activities were rather high(P<0.01). The optimum inoculum size of CMCase, Cotton lyase andβ-glucosidase were 3%, 7% and 7%respectively; The best temperature were 30℃, 28℃and 30℃; The best primary pH was 5.5; the optimum ferment time was 96h, 72h and 96h respectively.2. Degenerate primers were designed according to the Penicillium cellulase CBHⅠgene amino acid sequences that landed on NCBI, the goose origin Penicillium oxalicum Currie & Thom F67 CBHⅠgene fragment which was long 1059bp was cloned by RT-PCR(EU574736). The result of Blast analysis showed that this sequence had the highest homology with the gene of Penicillium janthinellum which could reach 84%. Then, arbitrary degenerate Primes and specific primers were designed on the basis of the CBHⅠgene fragment, the cDNA of Penicillium oxalicum Currie & Thom F67 was used as template to clone the 3 'and 5' flanking sequences by the improved thermal asymmetric interlaced PCR. The sequence analysis showed that the length of 3 'and 5' flanking sequences were 602bp and 728bp respectively, which contained start codon ATG and termination codon TAA.3. Specific primers were designed according to the 3 'and 5' flanking sequences of Penicillium oxalicum Currie & Thom F67 and the restriction sites EcoRⅠ/NotⅠwere introduced to clone the full-length of CBHⅠof 1638bp(EU727171). The gene encoded 545 amino acids and a stop codon TAA. The positive clones plasmid were double digested by EcoRⅠ/ NotⅠto connect with the secretory expression vector pPIC9K which was also double digested by EcoRⅠ/ NotⅠ, the identification of PCR and restriction enzyme digestion showed that the constructing of eukaryotic expression vector was sucessuful which was named pPIC9K-CBHⅠ.4. The bioinformatics analysis of Penicillium oxalicum Currie & Thom F67 CBHⅠgene amino acid sequence showed that the molecular weight and isoelectric point of it was 56.8kD and 4.9 respectively, the first 26 amino acids may be a signal peptide sequence with a rather strong hydrophobicity. There were 43 phosphorylation modification sites in the sequence, including 16 serine sites, 18 threonine sites and nine tyrosine sites. The result of structure and function projections showed that the structure and function domain of the protein consisted of three components, which were catalytic domain (aa30-461), convergence zone (aa462-512) and cellulose-binding domain (aa513-545) of glycosylation No.7 hydrolase family individually. Analysis of subcellular localization showed that there was about 55.6% protein existed in extracellular. The result of Blast analysis showed that this sequence had the highest homology with the gene of Penicillium janthinellum (X59054.1) which could reach 76%.