Senthil Kumar. M^1,, Selvam. K², Singaravel. R³, Krishnaveni. N⁴

A unique novel extracelluar glutaminase free L-asparaginase obtained from marine Streptomyces radiopugnans MS1 is an important therapeutic enzyme used in the treatment of Hepatocellular carcinoma. A full-length gene of L-asparaginase was cloned from Streptomyces radiopugnans MS1 and over expressed in Escherichia coli BL21 (DE3). The recombinant L-asparaginase from E.coli was purified by Model 491 Prep Cell which gave 136.8% higher yield with final specific activity of 742.8 IU/mg than the indigenous L-asparaginase from S. radiopugnans MS1. The molecular weight of L-asparaginase was found to be approximately 33.3 kDa by SDS–PAGE analysis. L-asparaginase was tested for its efficacy and cytotoxicity to HepG2 cell line with IC₅₀ 0.45 IU/mg and selectivity index (SI) at 10.8 which is about eleven time’s higher selectivity over the lymphocyte cells. HepG2 cells were the most sensitive, showing apoptosis with a higher incidence subsequent to L-asparaginase treatment. A multiplexed flow cytometric bead-based assay to analyze the release of cytokines and quantitative real-time PCR (qRT-PCR) to evaluate gene expression were performed. The obtained cytokine pattern showed that, at the increasing rate of two molecules concentrations, two pro-inflammatory cytokines such as VEGF and IL-8 were decreased whereas the anti-inflammatory cytokine such as IL-4 and IL-10 were increased. This is the first report of the cloning and functional expression of a glutaminase free L-asparaginase gene from noval marine Actinomycetes species. This study has endowed confirmation for the mechanism of L-asparaginase anticancer activity against Hepatocellular carcinoma which was compared with the commercial L-asparaginase. Results indicated that L-asparaginase could be utilized as an effective influential substance for cancer treatment.

glutaminase free L-asparaginase, Streptomyces radiopugnans MS1, HepG2 cells and cytokines