Abstract

omega-Transaminases are crucial biocatalysts for the asymmetric synthesis of chiral amines; however, their industrial application is often limited by insufficient stability at high concentrations of organic solvents. In this study, recombinant transaminase from Vibrio fluvialis (Vfat) was directly immobilized from crude cell lysates onto three supports: glyoxyl-functionalized silica, aminopropyl-functionalized silica, and glyoxyl-activated agarose. These systems were systematically evaluated in media containing high fractions of organic solvents (50-60% v/v). Support functionalization and enzyme deposition were confirmed by DRIFT-FTIR and SDS-PAGE, while immobilization kinetics and activity-based parameters revealed strong support-dependent effects. Glyoxyl-activated agarose enabled multipoint covalent immobilization, yielding the highest global activity recovery (IYa > 94%; GYa > 36%). This immobilization mode enhances structural rigidity and limits solvent-induced denaturation. Consequently, this support was selected for stability studies. Immobilized Vfat retained similar to 55% residual activity after 48 h in DMSO (50-60% v/v), whereas the soluble enzyme was fully inactivated (RA approximate to 0%). The high tolerance toward DMSO highlights the effectiveness of covalent immobilization in preserving enzyme structure. Overall, support chemistry and immobilization strategy critically define Vfat performance, with glyoxyl-activated agarose emerging as a robust platform for solvent-intensive biocatalysis.