Heterologous expression and molecular modelling of L-asparaginase from Bacillus subtilis ETMC-2.

Bacterial L-asparaginase is the key therapeutic enzyme in cancer therapy and is also witnessing demand as a food processing aid. In this study, L-asparaginase of newly isolated Bacillus subtilis ETMC-2 was cloned and over-expressed in Escherichia coli as an active soluble protein using ligation independent cloning strategy. The molecular mass was estimated to be 40 kDa and was optimally active at 50 °C. Zymography revealed that the enzyme was active in homo-tetramer state (~160 KDa). The encoded protein after BLASTp analysis on NCBI showed 99.73% similarity with L-ASNase that of Bacillus sp. Physico-chemical properties were predicted using Protparam leading to categorization of the enzyme as a stable protein with an instability index (II) of 19.02. The calculated aliphatic index (85.44) indicated the high thermal stability of the protein with GRAVY value of -0.317. Protein-Ligand docking revealed that the residues Thr, Thr, and Asp were fundamental in protein-ligand complexation. After homology modelling, model validation was performed using Ramachandran plot, VERIFY3D, and RMSD. The paper describes cloning, heterologous expression, catalytic characteristics and physico-chemical properties of the type II B. subtilis L-ASNase.