Screening Cellulose Decomposition Microorganisms And The Expression Of Celluloses Gene

Cellulose, a linear polymer ofβ-1,4-linked anhydrous glucose residues, is the most abundant biopolymer on the earth. Owing to the shortage of efficient techniques for cellulose degradation, limited success have been achieved on the utilization of cellulose. In nature, cellulose is degraded mainly by microorganisms, which possess a series of cellulose-hydrolyzing enzymes including cellobiohydrolases (CBHs; EC 3.2.1.91), endoglucanases (EGs; EC 3.2.1.4), andβ-glucosidases (EC 3.2.1.21). However, due to the low activities of commercial cellulase, degradation process becomes cost effective. Therefore, the attempts for screening new cellulase producing strains and purified new cellulase with specified characters were never stopped recently.In current study, we initially established a suitable screening model, and apply it to screen cellulose degradation fungal. Total nine fungus were isolated from the deep well earth sample and two of them demonstrated higher cellulase activities based on the results generated from filter paper degradation experiment and cellulose-azure degradation experiment.18S rRNA sequences suggested these two strains belonged to Penicillium purpuroum and Aspergillus fumigatus.Cellulose degradation activities were measured with CMC and filter paper as substrates. The endoglucanse activities of the two strains were 1.06IU and 0.81IU, whiles exoglucanse were 0.71IU and 0.19IU, respectively. The optimal temperature for cellobiohydrolase produced from A. fumigatus was 70℃, and the optimal pH was 3 to 4. In Subsequences we cloned cellobiohydrolase gene from Aspergillus fumigatus using the technicque of splicing by overlapping extension. then expressed in E.coli BL21 by pET28(a). SDS PAGE demonstrated the molecular weight is 52KDa. In addition, the purified cellobiohydrolase showed cellobiohydrolase activity at 70℃ when PNPC was used as substrate.