Cloning, Expression, Characterization and in silico studies of l-asparaginase from Vibrio sp. (GBPx3)

Abstract

l-asparaginase is a critical therapeutic enzyme for treating acute lymphoblastic leukemia (ALL), a common childhood malignancy. In this study, the l-asparaginase coding sequence from halophilic Vibrio sp. (GBPx3) was cloned, expressed in Escherichia coli, and characterized. The enzyme exhibited a molecular weight of 39.2 kDa and demonstrated a K_m of 4.517 mM, k_cat of 2.88 1/s, and V_max of 0.1055 μmol/min, reflecting high specificity for l-asparagine and minimal activity (0.4 %) toward l-glutamine. Optimal activity was observed at physiological conditions (37 °C, pH 7.5 and 125–150 mM NaCl), consistent with human serum osmolality. The half-life of the enzyme was 2.64 h in human serum at 37 °C that is longer than the half-life reported for E. coli l-asparaginase. Additionally, the enzyme had no toxic impact on human umbilical vein endothelial cells (HUVEC) and human erythrocytes. The recombinant l-asparaginase was predicted to be 29.3 % helix, 35.6 % turns, and 35.1 % random by circular dichroism spectroscopy. AlphaFold predicted a 3D structure with promising validation scores. The molecular docking study showed that Thr14, Ser60, Thr91, and Asp92 are putative active site residues, with a negative binding energy of −4.5 kJ/mol for the substrate-enzyme interaction. The enzyme's low immunogenicity, high serum stability, and reduced glutaminase activity highlight its potential as a safer therapeutic alternative. Future experiments and protein engineering studies are needed to explore enzyme's in vivo efficacy and improve its clinical effectiveness.