Cloning of Bacillus subtilis RSKK243 Bifunctional Xylanase Gene in E. coli and B. subtilis and Enzyme Characterization


Abstract: A bifunctional gene xylanase of B. subtilis RSKK243 encoding detectable beta(1,4) glucanase (CMCase) activity in addition to a high level of main xylanase activity was cloned and expressed in Escherichia coli with pUC18 plasmid and in various Bacillus subtilis strains with pUB110 vector. For enzymatic analysis and plasmid isolation, the gene was also expressed in xylanase and beta glucanase negative B.subtilis YB886 strain with pUB110 vector. The pUB110 with bifunctional xylanase gene was transferred into B. subtilis RSKK243 for maximizing the enzyme yield, as well as into other RSKK strains to create novel strains with multiple enzyme capacities. Recombinant strains were compared with their original counterparts with respect to their enzyme yield and multiple enzyme activities. In addition, molecular size determination and zymogram analysis were performed on SDS-PAGE and SDS-CMC-PAGE and SDS-Xylan-PAGE gels. The temperature and optimum pH of the enzyme were determined to be 50ºC and 5 respectively. The enzyme was totally inactivated when it was exposed to a temperature of 90ºC for 15 minutes.

Keywords: Bacillus subtilis, xylanase, beta-glucanase, gene cloning

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