Cloning,Expression And Analysis Of AprE Protein Gene From Bacillus Licheniformis

Bacillus licheniformis is one of the most important strain of protease production, and has very obvious advantage in industrial applications. But its protease system is still unclear now, further analysis of its protease system will be useful to obtain the genetical strains which meets the requirements of the industrial production. Alkaline protease from B.licheniformis is an important protease type in the industry. Improving its activity and stability under alkaline pH conditions can broaden the range of applications in detergent industry.In this study, protease WprA, Epr and AprE from B. licheniformis B186 and 2709 were expressed in Bacillus subtilis WB600. The AprE gene was found to be one of the major extracellular proteases of B. licheniformis, and the enzyme was the main object of this study.The protease AprE2709 from B. licheniformis 2709 and AprE209 from B.licheniformis B186 were chosen, and the encoding genes were successfully cloned and expressed in B. subtilis WB600. The recombinant plasmids were transformed into B.licheniformis Z113 by electroporation, generating the recombinant bacteriums Z113(pHY-AprE2709) and Z113(pHY-AprE209).Recombinant alkaline proteases AprE2709 and AprE209 were then purified to apparent homogeneity from the supernatant of the recombinant bacteriums using in exchange chromatography and filtration chromatography. The optimum pH and temperature of recombinant enzyme AprE209 were 11.0 and 50 ?, respectively. This recombinant enzyme also retained high activities at temperatures of 30-37 ? or pH 12.0. AprE2709 show the best enzyme activity at pH 10.0 and 50 ?, respectively. This recombinant enzyme also retained high activities at temperatures of 40-50 ? or pH 12.0. Furthermore,the mechanism of alkali-tolerance of AprE209 and AprE2709 were compared and analyzed by bioinformatics tools.