Production, optimization, purification, characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate.

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5-7.0 and 35 °C after 28 hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78 U/mg and its molecular weight about 54.8 kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40 °C and retained about 90% activity for 1 hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.

Purification and biotechnological applications of L-asparaginase from newly isolated Bacillus halotolerans OHEM18 as antitumor and antioxidant agent.

In the present study, a new bacterial strain, Bacillus halotolerans OHEM18 was significantly found to produce extracellular L-asparaginase. L-asparaginase was purified using ammonium sulfate precipitation and QFF column to 3.84-fold with specific activity of 215.33 U/mg and its molecular mass was assessed to be 41.5 kDa. Maximum enzyme activity was determined at pH 8.2 and 40 °C and with retaining 70% of its activity after incubation for 1 h at 50 °C. Km and Vmax values were determined to be 0.0047 M and 92.74, respectively. Cytotoxicity test indicated a significant safety of L-asparaginase on Vero cells with selectivity against leukemia, breast cancer and hepatoma cells. NFS-60 cells was the most sensitive tumor cells to L-asparaginase with IC50 of 10.29 µg/ml and selectivity index of 30.61. This selectivity was recognized to be an apoptosis-dependent mechanism proven via cell cycle arrest in sub-G1 phase and fragmentation of genomic DNA. L-asparaginase showed antioxidant activity against both DPPH and ABTS radicals with IC50 values of 64.07 and 177.1 mg/ml, respectively. These competitive advantage of bacterial L-asparaginase over than other sources is that it might be produced in large amounts through production in large-scale biofermenters, which decreases costs, besides having a sustainable bacterial source. Our findings established that the potent cytotoxic effect of L-asparaginase isolated from B. halotolerans OHEM18 may be a promise candidate for further medicinal applications as an antioxidant and antitumor drug.Communicated by Ramaswamy H. Sarma.