Single step purification and characterization of a thermostable and calcium independent α-amylase from Bacillus amyloliquifaciens TSWK1-1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India)

A thermophilic bacteria, identified and designated as Bacillus amyloliquifaciens TSWK1-1 (16S rRNA gene sequence, GenBank: GQ121033), was isolated from a hot water reservoir located at Tulsi Shyam, Gujarat, India. The optimum temperature and pH for amylase production were 50 °C and 7.0, respectively. The crude enzyme was partially purified by ammonium sulphate fractionation followed by dialysis. However, single step purification was achieved on Phenyl Sepharose 6FF affinity column with 45.71% yield, 8000 U/mg specific activity and 13.33 fold purification. The molecular weight of the purified α-amylase was 43 kD. The optimal temperature and pH for amylase activity were 70 °C and 7.0, respectively; however, the purified amylase was stable at broad temperature and pH range. The purified amylase did not require Ca++ and K+; however, it was moderately affected by Mg++ and Cu++ and significantly inhibited by Na+ and Fe++. The amylase was highly thermostable and remained active for 24 h at 60 °C, for 12 h at 70 °C and up to 3 h even at 90 °C. Other unique features of the enzyme were calcium independent nature and resistance against chemical denaturation by Urea and Guanidine-HCl. The data on the enzymatic stability at different levels of purity would add significantly to the knowledge of amylases.