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 hours 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.