SOLVENT-FREE ENZYMATIC BIODIESEL PRODUCTION USING FREE AND EVERSA® TRANSFORM 2.0 LIPASE IMMOBILIZED IN A PIL/LIGNIN-RICH SUGARCANE PRETREATMENT LIQUOR MATRIX

Enzymatic transesterification is a promising alternative for biodiesel production because it operates under mild conditions, reduces soap formation, and improves product selectivity. This study evaluated solvent-free fatty acid ethyl ester (FAEE) production from refined soybean oil using Eversa® Transform 2.0 lipase in free form and immobilized in Ca-alginate beads containing a sugarcane-straw pretreatment liquor enriched with the protic ionic liquid monoethanolamine acetate ([Mea][Ac]) and lignin. The immobilized biocatalyst was prepared with 2% (w/v) sodium alginate, 10% (w/w) lipase, 0.5% (w/w) [Mea][Ac], and 0.05% (w/w) lignin, and protein was quantified by the Bradford method. Soybean-oil fatty acids were characterized by gas chromatography using an Agilent 68650 instrument equipped with a capillary column. Biodiesel reactions were conducted in 10 mL Erlenmeyer flasks using free or immobilized enzyme corresponding to 1 mg of protein, at 40 °C and 350 rpm for 48 h. After centrifugation, FAEE, triacylglycerols, diacylglycerols, monoacylglycerols, and free fatty acids were quantified by high-performance size-exclusion chromatography using a Shimadzu 20AT system, a Phenomenex size-exclusion column, and a refractive-index detector. Both biocatalyst configurations promoted substantial conversion. The free enzyme achieved a biodiesel yield of 90.55%, whereas the immobilized enzyme reached 80%. The higher yield obtained with the free lipase indicates more effective enzyme-substrate contact in the solvent-free medium. The moderate reduction after immobilization is consistent with mass-transfer limitations and microenvironmental effects within the alginate matrix; however, the immobilized system maintained high catalytic activity and offers easier separation and potential reuse. Therefore, the PIL/lignin-rich pretreatment liquor can function as a viable, value-added component of an immobilization matrix, integrating a biorefinery-derived stream into biodiesel production. The authors acknowledge FAPESP (2022/01747-1), CAPES (Finance Code 001), CNPq (313579/2025-3 and 421662/2025-5), and FAEPEX/UNICAMP (519.287-2604/23) for financial support.