Thermostable -amylase from natural variants of Bacillus spp. prevalent in eastern Himalayan Range

Abstract

North Eastern Region of India in the eastern Himalayan Range has a rich diversity of microbial flora which has remained unexplored. It is thus, an ideal place to explore the rich and complex microbial diversity for the isolation of novel α-amylase enzyme. In the present study, various bacterial strains were isolated from soils of North-Eastern region of India spread over diverse habitats ranging from an altitude of 24 to 3855 msl. Thirty bacterial isolates were screened for α-amylase production by plate assay method. Sixteen such strains were found to be positive for amylase production on starch agar medium. The amylase activity was assayed by DNSA method at different time intervals (10 - 60 min). Two bacterial strains (MK8 and MA9) were selected for further assays based on their higher enzyme activity. Based on the morphological, physiological and biochemical characteristics and phylogenetic position as determined by 16S ribosomal DNA gene sequencing, the bacterial strains MK8 and MA9 were identified as Bacillus cereus and Bacillus subtilisrespectively. The optimum activity in shake-flask cultures was seen between 30 - 40 min of incubation and the maximum enzyme production was achieved during the stationary phase (36 - 48 h) of the growth of the organism. Bacterial cultures in stationary phase were taken for enzyme activity assay and the incubation time was optimized at 30 minutes based on the optimal activity. The maximal α-amylase activity was achieved at 60oC, pH 7 and 80oC, pH 6 - 8 for MA9 and MK8 respectively. The amylase of MK8 strain remained stable between 30 and 80°C, whereas, in case of MA9 strain, the amylase was found to be stable between 30 and 60°C. This indicates that the enzyme produced by MA9 strain is moderately thermostable while that of the MK8 strain is highly thermostable. Ca2+ enhanced the α-amylase activity of the MA9 strain whereas in case of MK8 strain, addition of Ca2+ did not show any significant increase in amylase activity. Pb2+ had higher inhibition of enzyme activity compared to Cd2+, Cr3+ and Zn2+. Nearly 100% was recovered with Cu2+ addition in case of MA9 strain but only 50% was recovered for MK8 strain.   Key words: α-amylase, screening, thermostable, optimization, Bacillus.