Isolation and characterization of gene encoding α-amylase from amylolytic bacteria associated with green algae in the coast side of Southern Sea, Yogyakarta, Indonesia
Almatin Puspa Dewi1, Endah Retnaningrum1, Langkah Sembiring1, Yekti Asih Purwestri1*
Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta, INDONESIA 55281
Abstract
Gene encoding α-amylase from amylolytic bacteria HS32 associated with green algae has been isolated and cloned into E.coli DH5α. Amylase gene was amplified using Ami-F and Ami-R primers resulting in 1.6 kb length. The PCR product was cloned into zero blunt TOPO cloning vector, followed by transformation to E.coli DH5α competent cell. The selection of positive transformants were performed using Kanamycin selection medium. Analysis of positive transformed cells by colony PCR method using primer Ami-F and Ami-R produced 1.6 kb and using M13 universal primers produced 1.8 kb length. Restriction analysis of plasmid transformants by using restriction enzymes NotI and BamHI produced two bands, they were 3.5 kb (cloning vector) and 1.6 kb (insert gene). The BLAST sequence indicated that Bacillus sp. HS32 had 96% similarity with amylase gene of Bacillus cereus NC7401. This result open the possibility to characterize the properties of this α-amylase. Further research in genetic engineering for amylase production in E.coli expression vector need to be done.
Keywords: α-amylase, algae, cloning, BLAST, Bacillus cereus NC7401
Topic: Environmentally Sustainable Agriculture