<![CDATA[Pseudomonas cepacia lipase was immobilized on low-density polyglutaraldehyde–styrene-coated expandable polystyrene beads (PGlu–STY/EPS) and applied as a reusable biocatalyst for biodiesel production from soybean oil via ethanolysis. The catalyst system was developed through a progressive catalyst-engineering strategy involving floating-support design, support-density engineering, particle-size optimization, and coating-morphology refinement to improve catalyst distribution, interfacial accessibility, and operational stability. The immobilized lipase exhibited a protein-loading yield of 71.81% and a catalytic activity of 26.12 U g⁻¹ support. Smaller EPS particles and optimized PGlu–STY coating conditions improved enzyme-immobilization efficiency and catalytic performance by enhancing substrate accessibility and reducing diffusion limitations. Under optimized transesterification conditions—an oil-to-ethanol molar ratio of 1:5, a temperature of 40 °C, a reaction time of 24 h, and absolute ethanol—the maximum fatty acid ethyl ester (FAEE) conversion reached 92.8%. Biodiesel conversion was quantified using ¹H NMR spectroscopy. The immobilized catalyst retained substantial catalytic activity over more than 10 consecutive reaction cycles, indicating favorable operational stability and reusability. FT-IR analysis suggested successful covalent immobilization through Schiff-base interactions between aldehyde groups on the support and amino groups on the enzyme. The floating, low-density EPS architecture may improve catalyst distribution in the heterogeneous oil–alcohol reaction medium and reduce unfavorable sedimentation in glycerol-rich regions, thereby enhancing interfacial transesterification. Although advanced characterization techniques such as BET, XPS, and GC–MS were not available in the present study, the combined catalytic and morphological results demonstrate that PGlu–STY/EPS is a potentially useful support for reusable immobilized-lipase systems in enzymatic transesterification.]]>
