DETERMINATION AND OPTIMIZATION OF CRUDE EXTRACELLULAR AMYLASE EXTRACTED FROM DIFFERENT BACILLUS SPECIES

Abstract

Amylases are starch hydrolyzing enzymes and have commercial applications in different industries, such as food, fermentation, detergent and pharmaceuticals. In the current study amylases were extracted from bacteria found in kitchen wastes and rice fields. Isolates with maximum starch hydrolysis determined by the clear zones formation against blue background were selected and examined for further extracellular amylase activity. 16S rRNA gene sequencing confirmed these isolates as Bacillus subtilis, Bacillus sp. and Lysinibacillus sp. Highest dextrizing activity (83%)f and starch hydrolysis ratio (2.8) were shown by Bacillus subtilis G3. Furthermore optimum amylase activity was also checked at different temperatures, pH, media composition and substrate concentration. Optimum reaction temperature and pH for crude amylase activity from Bacillus subtilis G3 was 60°C -75°C and 9pH in minimal media supplemented with 0.1% soluble starch. Thin layer chromatography and high performance liquid chromatography were carried out to investigate the starch hydrolyzed products (glucose, maltose and maltosaccharides) produced by amylolytic action of amylase. In addition to it molecular weight of amylase was found to be about 68kDa as revealed by SDS PAGE analysis after acetone precipitation. These bacteria could be genetically manipulated for large scale enzyme production which can be ultimately used for commercial purposes.