title: Extracellular L-asparaginase from a protease-deficient Bacillus aryabhattai ITBHU02: purification, biochemical characterization, and evaluation of antineoplastic activity in vitro.
abstract: An extracellular L-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg(-1). The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified L-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K m, V max, and k cat values of the enzyme were 0.257 mM, 1.537 U μg(-1), and 993.93 s(-1), respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α + β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified L-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the L-asparaginase has significant antineoplastic properties.
authors.affiliation: School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005, India.
authors.forename: Yogendra
authors.initials: Y
authors.lastname: Singh
authors.forename: Ravi Kumar
authors.initials: RK
authors.lastname: Gundampati
authors.forename: Medicherla V
authors.initials: MV
authors.lastname: Jagannadham
authors.forename: S K
authors.initials: SK
authors.lastname: Srivastava
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D001215:Asparaginase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001407:Bacillus / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D045744:Cell Line, Tumor; D004795:Enzyme Stability; D005110:Extracellular Space / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010447:Peptide Hydrolases / Q000172:deficiency*; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000970:Antineoplastic Agents; D010447:Peptide Hydrolases; D001215:Asparaginase