Bacillus tequilensis PV9W, a marine bacterial isolate obtained from Gulf of Mannar, Rameswaram, India, produced glutaminase free L-asparaginase which was purified to homogeneity with a significant increase (13 fold) in specific activity. The apparent K_m (0.045 ± 0.013 mM) and V_max (7.465 ± 0.372 μmol ml⁻¹ min⁻¹) values of this purified L-asparaginase was identified and it was found that the maximum activity of the L-asparaginase was at a pH 8.5 and a temperature of 35 °C. The enzyme is a mixed α/β protein and the influence of different effectors were documented. The purified L-asparaginase had effective acrylamide degradation activity (6 IU per ml) and cytotoxic activity against HeLa cell lines with an IC₅₀ of 0.036 ± 0.009 IU per ml. Furthermore, the purified enzyme showed p53 dependent G₂ arrest in HeLa cells analyzed by FACS and was hemocompatible. Thus, this study highlights the marine isolate PV9W as a potential source for glutaminase free L-asparaginase with industrial as well as pharmaceutical applications. This study also paves a way for a possible therapeutic drug with the least amount of side effects.