Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S : Cloning and characterization of the lipase(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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A thermophilic microorganism, SBS-4S, was isolated from a hot spring located in Gilgit, Northern Areas of Pakistan. It was found to be an aerobic, gram-positive, rod-shaped, thermophilic bacterium that grew on various sugars, carboxylic acids and hydrocarbons at temperatures between 45℃ and 75℃. Complete 16S rRNA gene sequence of the microorganism exhibited homology to various species of genus Geobacillus. A highest homology of 99.8% was found with Geobacillus kaustophilus. A partial (0.7kbp) chaperonin gene sequence also showed a highest homology of 99.4% to that of G. kaustophilus whereas biochemical characteristics of the microorganism were similar to Geobacillus uzenensis. Based on biochemical characterization, 16S rRNA and chaperonin gene sequences, we identified SBS-4S as a strain of genus Geobacillus. Strain SBS-4S produced several extracellular enzymes including amylase, protease and lipase. The lipase encoding gene was cloned, expressed in Escherichia coli and the gene product was characterized. The recombinant lipase was optimally active at 60℃ with stability at wide pH range (6-12). The enzyme activity was enhanced remarkably in the presence of Ca^<2+>. The K_m and the V_<max> for the hydrolysis of p-nitrophenyl acetate were 3.8mM and 2273μmol min^<-1>mg^<-1>, respectively. The ability of the recombinant enzyme to be stable at a wide pH range makes it a potential candidate for use in industry.