Cloning Of A Novel Cellulase Gene From Bacill Us Pumilus Newly Isolated From Tibetan Swine

Tibetan swine is a grass-type oriented local species which is adaptive to be raised on theQinghai-Tibet plateau. It is year-round grazing and mainly lives on grass. Long term naturalselection makes it cold and disease resistat, roughage tolerant and meat tasty. To research themechanisms that lead to roughage tolerant, BHM medium was applied to select and screencellulolytic microbes and from which commercial cellulase gene was cloned. This researchcan lay foundations for research and utilization of cellulolytic microbes and cellulases. Themain conclusions are as follows:1. Targeted bacteria was screened and identified by Gram staining with16S rDNA partialsequence alignment. One of the selected ones designated as Bacillus pumilus BY-1with highenzyme activity was selected for further research.2. The maximum enzyme activity was0.9216IU/mL. The optimum temperature of thecrude enzyme was55℃. When the temperature reached60℃and70℃, it retained60%and30%activity respectively. The optimum pH of the crude enzyme was7.45. Furtherdetermination showed it was pH resistant, it retained more than70%of its activity when thepH was between5.15and8.26, it still reached50%activity even when the pH was9.08.3. Taking bglC as reference to design primers, a cellulase gene bglC-BY wassuccessfully amplified from the isolated bacteria. It is a new gene of1851bp with aGH9/CBM3molecular organization4. Heterogeneous expression was conducted in E.coli and confirmed by SDS-PAGE andenzyme activity detection. SDS-PAGE verified the successful expression of bglC-BY with amass of about70kDa and the enzyme activity was0.5413IU/mL. Thus, we provided a newstrain and a new endoglucanase gene with great potential for industrial processes.The results of this research were important to reveal the mechanisms of roughage,cultivate the herbivorous swine breed and develop grain saving swine industry.