Biocatalyst engineering of Thermomyces Lanuginosus lipase adsorbed on hydrophobic supports: Modulation of enzyme properties for ethanolysis of oil in solvent-free systems

Different immobilized biocatalysts of Thermomyces lanuginosus lipase (TLL) exhibited different properties for the ethanolysis of high oleic sunflower oil in solvent-free systems. TLL immobilized by interfacial adsorption on octadecyl (C-18) supports lost its 1,3-regioselectivity and produced more than 99% of ethyl esters. This reaction was influenced by mass-transfer limitations. TLL adsorbed on macroporous C-18 supports (616 A of pore diameter) was 10-fold more active than TLL adsorbed on mesoporous supports (100-200 A of pore diameter) in solvent-free systems. Both derivatives exhibited similar activity when working in hexane in the absence of diffusional limitations. In addition, TLL adsorbed on macroporous Purolite C-18 was 5-fold more stable than TLL adsorbed on mesoporous Sepabeads C-18. The stability of the best biocatalyst was 20-fold lower in anhydrous oil than in anhydrous hexane. Mild PEGylation of immobilized TLL greatly increased its stability in anhydrous hexane at 40 degrees C, fully preserving the activity after 20 days. In anhydrous oil at 40 degrees C, PEGylated TLL-Purolite C18 retained 65% of its initial activity after six days compared to 10% of the activity retained by the unmodified biocatalyst. Macroporous and highly hydrophobic supports (e. g., Purolite C-18) seem to be very useful to prepare optimal immobilized biocatalysts for ethanolysis of oils by TLL in solvent-free systems.