title: Purification and Characterization of an Extracellular Cholesterol Oxidase of Bacillus subtilis Isolated from Tiger Excreta.
abstract: A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (COX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of COX by manipulation of various physicochemical parameters. The extracellular COX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified COX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of ∼21 kDa protein) nature, organic solvent compatibility, and a half-life of ∼2 h at 37 °C. The bacterial COX exhibited ∼22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K m and V max value of COX of B. subtilis COX were found to be 3.25 mM and 2.17 μmol min ml(-1), respectively. The purified COX showed very little cytotoxicity associated with it.
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Shimla, 171 005, India. latahp@gmail.com.
authors.forename: Lata
authors.initials: L
authors.lastname: Kumari
authors.affiliation: Department of Biotechnology, Himachal Pradesh University, Shimla, 171 005, India. kanwarss2000@yahoo.com.
authors.forename: Shamsher S
authors.initials: SS
authors.lastname: Kanwar
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D002021:Buffers; D002460:Cell Line; D002474:Cell-Free System; D002789:Cholesterol Oxidase / Q000378:metabolism*; D002846:Chromatography, Affinity; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005243:Feces / Q000382:microbiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010802:Phylogeny; D046010:Tigers
chemical_list: D002021:Buffers; D003902:Detergents; D002789:Cholesterol Oxidase
keywords: Bacillus subtilis; Biochemical characterization; Extracellular cholesterol oxidase; Purification