Purification and characterisation of a novel broad spectrum anti-tumor L-glutaminase enzyme from marine Bacillus subtilis strain JK-79

L-glutaminase is a therapeutic enzyme used in the treatment of acute lymphoblastic leukemia (ALL). In this study, the extracellular L-glutaminase produced by Bacillus subtilis strain JK-79 was purified to homogeneity. The purified L-glutaminase has specific activity of 937 U mg−1 with KM and VMax value of 8 × 10-2 mM and 200 µM/ml min, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed it to have a molecular mass of 64 kDa. Further biochemical characterization revealed that the purified enzyme attained maximal activity at pH 7 and temperature 37°C. The enzyme was stable at 37°C for 60 min at physiologic-ph. The enzyme showed high specificity towards L-glutamine. The effect of metal ions on enzyme activity showed that NaCl, KCl and CaCl2 enhanced the activity while AgCl3 and CuCl2 strongly inhibited the activity of the enzyme. The in-vitro antioxidant activity of the enzyme was investigated and the IC50 value for DPPH and ABTS assay were 400 and 600 µg/ml, respectively. The purified enzyme showed cytotoxic activity against various Leukemic cell lines tested with IC50 value of 231, 480, and 500 µg/ml for K562, U932 and Jurkat cell lines, respectively. Interestingly, L-glutaminase also revealed cytotoxic effect on other cancer cell lines such as MCF-7, OV1063 and HCA 7 with IC50 value of 500, 526 and 750 µg/ml, respectively. Hence this enzyme from marine bacteria would possibly be an attractive candidate for further pharmaceutical use as a broad spectrum anti-tumor drug.   Key words: L-glutaminase, purification, characterization, anti-oxidant activity, anti-cancer activity.