title: 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).
abstract: 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 24h at 60 °C, for 12h at 70 °C and up to 3h 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.
authors.affiliation: Department of Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India.
authors.forename: B A
authors.initials: BA
authors.lastname: Kikani
authors.forename: S P
authors.initials: SP
authors.lastname: Singh
mesh_terms: D000645:Ammonium Sulfate; D001407:Bacillus / Q000737:chemistry*; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D045482:Hot Springs / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D007194:India; D007700:Kinetics; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000516:alpha-Amylases; D000645:Ammonium Sulfate