A novel thermostable class 3 l-asparaginase from Arthrobacter psychrolactophilus: Characterization and potency against MDA-MB-231 breast cancer cells.

In this study, a novel Class 3 l-asparaginase (ASNase) from Arthrobacter psychrolactophilus was successfully characterized. Belonging to the rarely reported Class 3 group, this enzyme was identified as a 70 kDa homodimer, consisting of subunits with a molecular weight of approximately 35 kDa. The enzyme exhibited optimal catalytic activity at pH 9.0 and 45 °C. Notably, Ap ASNase exhibited substantial thermal stability, with a half-life of 42.27 min at 60 °C and a melting temperature (Tm) of 50.15 °C, demonstrating considerable structural robustness despite originating from a psychrophilic microorganism. Kinetic analysis revealed Km of 2.738 mM, indicating the high substrate affinity recorded within Class 3, alongside a kcat and catalytic efficiency of 8035 min-1 and 2934.62 mM-1 min-1, respectively. A key therapeutic advantage of this enzyme is its remarkably low glutaminase activity (1.4%), which minimizes potential side effects. Furthermore, the enzyme showed potent cytotoxicity against MDA-MB-231 cancer cells with an IC50 value of 3.9 U/mL. These biochemical properties underscore the potential of this ASNase as a high-performance candidate for biomedical and pharmaceutical applications.