Amplification, Sequencing and Cloning of Iranian Native Bacillus subtilis Alpha-amylase Gene in Saccharomyces cerevisiae

Abstract

Background: Alpha-amylases are digestive enzymes which hydrolyze starch glycosidic bonds to glucose, maltose, maltotriose and dextrin which have diverse applications in a wide range of industries such as food, textile, paper, detergents representing approximately 30% of the world enzyme production. Objectives: In this study, the gene encoding the alpha-amylase enzyme of native isolated Bacillus subtilis was amplified with specific primers containing of NotI and AscI restriction sites by PCR and then sequenced. Purified PCR product and shuttle episomal vector p316TDH3 were cut by restriction enzymes and cloned into Escherichia coli and yeast hosts. Materials and Methods: The haploid auxotroph (ura3-) strain of S. cerevisiae and p316TDH3 were used as the host and vector for cloning and expression of the alpha amylase gene, respectively. In native Bacillus sp. the amyE gene without signal sequence was amplified with specific primers that introduced AscI and NotI restriction sites. After constructing the recombinant plasmid, it was transformed into E. coli competent cells. Then, colonies selection and confirmation were performed and the extracted plasmid was introduced to competent yeast cells using carrier sperm DNA. Recombinant yeast cells could grow on minimal media and produce extracellular enzyme. Results: The presence of alpha-amylase gene in recombinant bacteria was certificated by colony-PCR method. After extraction of recombinant vector from E. coli, the competent S. cerevisiae cells were transformed using polyethylene glycol and carrier sperm DNA. The recombinant yeast strains were screened by URA3 auxotrophic marker and analyzed for alpha-amylase gene existence. In the other hand, the amylase gene length of native B. subtilis was1887base pairs (bp) with an approximately93.65% similarity with standard bacterial strain. Conclusions: Based on this similarity and our bioinformatics evaluations, this mentioned alpha-amylase gene can be expressed in S. cerevisiae as extracellular enzyme.