L-asparaginase from the mangrove endophyte Penicillium citrinum MEF 455: a focus on cancer surveillance gene expression in tumor cell lines HL-60 and NCI-H 460.

Marine endophytic fungi serve as a valuable source of bioactive molecules, with growing applications in enzyme production. This study investigates the therapeutic potential of glutaminase- and urease-free Type II L-asparaginase derived from the mangrove endophyte Penicillium citrinum MEF 455 against neoplastic cells. Extracellular L-asparaginase production was done using Czapek Dox broth amended with L-asparagine and a 66 kDa molecular mass asparaginase could be observed. The specific activity of 41.6 U/mg with 5.8-fold purification was attained using DEAE cellulose and Sephacryl S-200 column. The kinetic study showed that the Km, Vmax_, and Kcat were 1.370 mM, 161.29 U/mL/min, and 1240.69/min, respectively. Purified L-asparaginase displayed optimal activity at 40 °C and pH 8, with a substrate concentration of 2.5 mM L-asparagine. Metallic ions like Na⁺, K⁺, Mg²⁺, Co²⁺, and Li⁺, improved asparaginase activity. The enzyme displayed strong anticancer potential with considerable reduction in the growth of HL-60, and NCI-H 460 cells with IC₅₀ values of 0.37 ± 0.225 U/mL and 0.39 ± 0.176 U/mL, respectively. Major cancer-controlling genes i.e. p53, caspase-3, caspase-9, NF-kB, Bax, and Rb1 were up-regulated. In contrast, anti-apoptotic i.e. Cox-2 and Bcl-2 were down-regulated on asparaginase treatment in Human cancer cell lines HL-60 and NCI-H 460. The experimental study demonstrates that Type II L-asparaginase produced from an endophytic fungal source, P. citrinum MEF 455, was free from glutaminase and urease activity, thereby minimizing associated immunogenic complications. In general, understanding the physicochemical properties and functionality of the enzyme highlights its potential as a promising antitumor candidate for therapeutic development and clinical applications.