Background: Microbial lipases are among the most industrially significant biocatalysts, finding applications in food processing, biodiesel synthesis, pharmaceutical manufacturing, and detergent formulation. Pseudomonas aeruginosa-derived lipases exhibit broad substrate specificity and thermostability, making them particularly attractive for bioprocess applications.

Objective: This study aimed to optimize lipase production from Pseudomonas aeruginosa ATCC 27853 using fed-batch fermentation strategies and evaluate four enzyme immobilization approaches to enhance catalytic stability and reusability.

Method: Five fermentation configurations (batch, three fed-batch variants at different temperatures, and continuous mode) were evaluated. Enzyme immobilization was performed on silica gel, chitosan beads, magnetic Fe₃O₄ nanoparticles, and polyurethane foam. Kinetic parameters (Km, Vmax, kcat) were determined using p-nitrophenyl palmitate as substrate. Statistical analysis used response surface methodology (RSM) and one-way ANOVA (SPSS v.27).

Key Results: Fed-batch fermentation at 30°C with pH 7.2 and 250 rpm agitation yielded maximum lipase activity (31.2 ± 2.1 U/mL), a 151.6% improvement over batch control. Magnetic nanoparticle cross-linked enzyme aggregates (CLEAs) demonstrated the highest thermal stability (t½ = 71.3 h) and reusability (22 cycles at >50% residual activity). Km values increased moderately upon immobilization (4.82 to 6.14 mM), indicating partial steric hindrance.