Production and characterization of a promising microbial-derived lipase enzyme targeting BCL-2 gene expression in hepatocellular carcinoma

Context and goal This study aimed to isolate and optimize a high-yield lipase-producing Pseudomonas aeruginosa strain from biological samples, enhance enzyme production through random mutagenesis, and evaluate its potential anticancer activity. Fifty-one biological samples (blood, urine, sputum, wound pus) were screened, and three isolates demonstrated significant lipase activity. The isolate with the highest activity, identified as P. aeruginosa (GenBank accession number PP436388), was subjected to ethidium bromide-induced mutagenesis, resulting in a two-fold increase in lipase activity (312 U/ml). Lipase production was optimized using submerged fermentation, with critical factors identified statistically as Tween 80, peptone, and substrate concentration. The enzyme was purified via ammonium sulfate precipitation and Sephadex G-100 chromatography, and its molecular weight (53 kDa) was confirmed by SDS-PAGE. Findings Optimal conditions for enzyme production included a pH of 9, temperature of 20 degrees C, and a 24-h incubation period. The partially purified enzyme exhibited high stability at pH values up to 10 and storage temperatures of 4 degrees C. Anticancer activity was evaluated using the MTT assay, revealing an IC50 of 78.21 U/ml against human hepatocellular carcinoma using HepG-2 cells, with no cytotoxicity observed against Vero cells. Flow cytometry confirmed that the enzyme's anticancer potential was mediated through apoptosis and necrosis. QRT-PCR data revealed that the expression of the Bcl-2 gene was significantly downregulated by 62% (P < 0.05) following the treatment of HepG-2 cells with the lipase enzyme. These findings suggest that lipase from P. aeruginosa holds promise as a novel therapeutic agent for hepatocellular carcinoma, addressing the limitations of current treatments.