Cloning Alpha-Amylase Gene from Iranian Indigenous Bacillus Licheniformis in Bacillus Subtilis
Majid MoghbeliBiology Department, Islamic Azad University, Damghn Branch, Damghan, Iran
Abstract: Amylase is one of the most used enzymes in industry. It is an extracellular enzyme and has allocated 20% of the enzyme marketing to itself. Amylases are widely used in several industrial processes such as food, wood, paper, detergent, and pharmaceutical production, and fermentation. Due to their high potential of amylase overexpression, Bacilli are considered as the most important and prevalently used as the producers of this enzyme. Thus cloning an amylase expressing gene in a well-known host such as Escherichia coli and Bacillus subtilis can aid the process of identification and production of new amylases and their functional improvement. Bacillus licheniformis genome was extracted and the amylase-coding gene was isolated and amplified using PCR with genome as the template and specific primers. The amplified amylase gene was transferred to T.vector which subsequently was digested by HindIII and BamHI. The digested fragment was ligated to the shuttle vector pMR12 and transferred into E. coli TOP10 cells. After confirmation of successful transformation of E. coli TOP10 cells with the recombinant plasmid, it was extracted and directly transferred into B. subtilis WB600. Alpha-amylase gene was cloned in pMR12 vector and B subtilis as the host and the presence of the gene fragment in kanamycin-resistant colonies was confirmed using enzymatic digestion and PCR. Amylase gene was successfully cloned according to the stability of the recombinant vector harboring this gene after several consequent subcultures.
Keywords: Alpha-amylase gene; Cloning; Bacillus licheniformis; Bacillus subtilis; PCR Back to TOC