Isolasi, Karakterisasi, dan Kloning Gen Penyandi α-Amilase Bakteri Halofil Moderat asal Bledug Kuwu

Abstract

A moderately halophilic bacteria, BK-05, was isolated from Bledug Kuwu, a saline terrestrial area at Central Java. Based on partial sequence of 16S-rRNA gene, the isolate was closely related to Halobacillus litoralis. This isolate showed amylolytic activity when grown on saline media [15% (w/v) NaCl] suplemented with starch. A pair of primer was designed based on the sequence of amyH gene from Halomonas meridiana and Pseudoalteromonas haloplanktis. PCR amplification using these primers showed three DNA bands with each size approximately 1.50, 1.00, and 0.75 kb. Partial DNA sequencing analysis based on its deduced protein sequence revealed that the 1.50 kb band was closely related to the sequence of metalloprotease from Bacillus subtilis (approximately 54.3% identity in 184 amino acid overlap). Southern hybridization analysis showed that the 1.50 kb fragment was located within a 4.0 kb fragment of BamHI, 4.8 kb of EcoRI, 4.3 kb of HindIII, and 4.0 kb of XhoI digestion of BK-05 genomic DNA, respectively.