Immobilization on Glyoxyl-agarose support (Gx) is one of the best strategies to stabilize enzymes. However, the strategy is difficult to apply at neutral pH when most enzymes are stable and, even when possible, produces labile derivatives. This work contributes to overcoming this hurdle through a strategy that combines solid-phase amination, presence of key additives, and derivative basification. To this end, aminated industrial lipases from Candida artarctica (CAL), Thermomyces lunuginosus (TLL), and the recombinant Geobacillus thermocatenulatus (BTL2) were immobilized on Gx for the first time at neutral pH using anthranilic acid (AA) or DTT as additives (immobilization yields >70%; recovered activities 37.5-76.7%). The spectroscopic evidence suggests nucleophilic catalysis and/or adsorption as the initial lipase immobilization events. Subsequent basification drastically increases the stability of BTL2-glyoxyl derivatives under harsh conditions (t1/2, from 2.1-54.5 h at 70 °C; from 10.2 h-140 h in 80% dioxane). The novel BTL2-derivatives were active and selective in fish oil hydrolysis (1.0-1.8 μmol of polyunsaturated fatty acids (PUFAs) min-1·g-1) whereas the selected TLL-derivative was as active and stable in biodiesel production (fatty ethyl esters, EE) as the commercial Novozyme®-435 after ten reaction cycles (~70% EE). Therefore, the potential of the proposed strategy in producing suitable biocatalysts for industrial processes was demonstrated.
Keywords: PUFAs (polyunsaturated fatty acids); biodiesel; covalent immobilization; glyoxyl–agarose; lipase; nucleophilic catalysis.