Biochemical and molecular insights on the bioactivity and binding interactions of Bacillus australimaris NJB19 L-asparaginase.

L-asparaginase, an antileukemic enzyme, is indispensable to the treatment of Acute Lymphoblastic Leukemia (ALL). However, the intrinsic glutaminase activity entails various side effects to the patients; thus, an improved version of the enzyme lacking glutaminase activity would be a requisite for effective treatment management of ALL. The present study highlights the biochemical and molecular characteristics of the recombinant glutaminase-free L-asparaginase from Bacillus australimaris NJB19 (BaAsp). Investigation of the active site architecture of the protein unraveled the binding interactions of BaAsp with its substrate. Comparative analysis of the L-asparaginase sequences revealed few substitutions of key amino acids in the BaAsp that could construe its substrate selectivity and specificity. The purified heterologously expressed protein (42 kDa) displayed maximum L-asparaginase activity at 35-40 °C and pH 8.5-9, with no observed L-glutaminase activity. The kinetic parameters, Km and Vmax, were determined as 45.6 µM and 0.16 µmoles min-1, respectively. Furthermore, in silico analysis revealed a conserved zinc-binding site in the protein, which is generally implicated in inhibiting the L-asparaginase activity. However, BaAsp was not inhibited by zinc at 1 mM concentration. Therefore, the findings provide insights on the biochemical and molecular details of BaAsp, which could be valuable in formulating it for alternate antileukemic drug therapy.