Expression, characterization and cytotoxicity of recombinant l-asparaginase II from Salmonella paratyphi cloned in Escherichia coli.

L-asparaginase is a remarkable antineoplastic enzyme used in medicine for the treatment of acute lymphoblastic leukemia (ALL) as well as in food industries. In this work, the L-asparaginase-II gene from Salmonella paratyphi was codon-optimized, cloned, and expressed in E. coli as a His-tag fusion protein. Then, using a two-step chromatographic procedure it was purified to homogeneity as confirmed by SDS-PAGE, which also showed its monomeric molecular weight to be 37 kDa. This recombinant L-asparaginase II from Salmonella paratyphi (recSalA) was optimally active at pH 7.0 and 40 °C temperature. It was highly specific for L-asparagine as a substrate, while its glutaminase activity was low. The specific activity was found to be 197 U/mg and the kinetics elements K, V, and k were determined to be 21 mM, 28 μM/min, and 39.6 S, respectively. Thermal stability was assessed using a spectrofluorometer and showed T value of 45 °C. The in-vitro effects of recombinant asparaginase on three different human cancerous cell lines (MCF7, A549 and Hep-2) by MTT assay showed remarkable anti-proliferative activity. Moreover, recSalA exhibited significant morphological changes in cancer cells and IC values ranged from 28 to 45.5 μg/ml for tested cell lines. To investigate the binding mechanism of SalA, both substrates L-asparagine and l-glutamine were docked with the protein and the binding energy was calculated to be -4.2 kcal mol and - 4.4 kcal mol, respectively. In summary, recSalA has significant efficacy as an anticancer agent with potential implications in oncology while its in-vivo validation needs further investigation.