L-asparaginase from the mangrove endophyte <i>Penicillium citrinum</i> 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 <i>Penicillium citrinum</i> 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 <i>Km</i>, <i>Vmax_,</i> and <i>Kcat</i> 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, <i>P. citrinum</i> 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.