Extracellular L-asparaginase from a protease-deficient Bacillus aryabhattai ITBHU02: purification, biochemical characterization, and evaluation of antineoplastic activity in vitro.

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.