Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi...

Microbial L-asparaginase (ASNase) is an important anticancer agent that is used extensively worldwide. In this study, 40 bacterial isolates were obtained from the Red Sea of Saudi Arabia and screened for ASNase production using a qualitative rapid plate assay, 28 of which were producing large L-asparagine hydrolysis zones. The ASNase production of the immobilized bacterial cells was more favorable than that of freely suspended cells. A promising isolate, KKU-KH14, was identified by 16S rRNA gene sequencing as Bacillus licheniformis. Maximal ASNase production was achieved using an incubation period of 72 h, with an optimum of pH 6.5, an incubation temperature of 37 °C, an agitation rate 250 rpm, and with glucose and (NH₄)₂SO₄ used as the carbon and nitrogen sources, respectively. The glutaminase activity was not detected in the ASNase preparations. The purified ASNase showed a final specific activity of 36.08 U/mg, and the molecular weight was found to be 37 kDa by SDS-PAGE analysis. The maximum activity and stability of the purified enzyme occurred at pH values of 7.5 and 8.5, respectively, with maximum activity at 37 °C and complete thermal stability at 70 °C for 1 h. The K_m and V_max values of the purified enzyme were 0.049995 M and of 45.45 μmol/ml/min, respectively. The anticancer activity of the purified ASNase showed significant toxic activity toward HepG-2 cells (IC₅₀ 11.66 µg/mL), which was greater than that observed against MCF-7 (IC₅₀ 14.55 µg/mL) and HCT-116 cells (IC₅₀ 17.02 µg/mL). The results demonstrated that the Red Sea is a promising biological reservoir, as shown by the isolation of B. licheniformis, which produces a glutaminase free ASNase and may be a potential candidate for further pharmaceutical use as an anticancer drug.