EFFECT OF CROSSLINKING ON FUNGAL CELLS IMMOBILIZED ON POLYETHYLENE TEREPHTHALATE GLYCOL (PETG) 3D SUPPORTS FOR FRUCTOOLIGOSACCHARIDES PRODUCTION

The crosslinking of catalytic biomass immobilized on porous supports can provide enhanced operational stability, enabling the use of these biocatalysts in long operating cycles, which is essential for industrial applications. In this context, the immobilization of Aspergillus oryzae IPT-301 cells, containing adhered fructosyltransferase enzymes, on polymeric 3D supports is being investigated for fructooligosaccharides (FOS) production via transfructosylation sucrose, a process of commercial interest due to its health benefits. This study aimed  to evaluate the effect of crosslinking on A. oryzae IPT-301 cells immobilized on cubic polyethylene terephthalate glycol (PETG) 3D supports during the transfructosylation of sucrose. Crosslinking was performed by placing the biocatalyst in a medium composed of 134.7 mL of 0.2 mol L^-1 (pH 7,9) tris-acetate buffer and 12.6 mL of 25 % (v v^-1) glutaraldehyde solution at 200 rpm and 25 °C for 45 minutes. And the end of the reaction, it was deactivated with 1.5 mL of 100 g L^-1 NaBH₄ solution, dissolved in 1 x 10^-3 mol L^-1 NaOH, for 30 minutes. Operational stability, for both the crosslinked and non-crosslinked biocatalyst, was evaluated over consecutive cycles of enzymatic reactions. One unit of transfructosylation activity (A_T) was defined as the  amount of enzyme capable of transferring 1 µmol of fructose per minute. Using a medium composed of 11.1 mL of 480 g L^-1 P.A. sucrose solution and 3.6 mL of 0.2 mol L^-1 tris-acetate buffer  (pH 5,5), the reaction was carried out for 1h at 200 rpm and 50°C. After each cycle, the biocatalysts were vacuum-filtered and washed with distilled water. Experiments were performed in triplicate. Fifteen A_T cycles were compared, and the results were analyzed using the ANOVA statistical test (significance level of 5%) for both biocatalysts. For the non-crosslinked biocatalyst, maximum A_T of 1013.70 ± 9.75 U g^-1 was obtained during the first eight cycles, after which (cycle 9 to 15) decreased by 82.34%. For the crosslinked biocatalyst, a maximum A_T of 783.63 ± 134.88 U g^-1 was obtained during the first eight cycles, and from cycle 9 to 15, there was a 74.15% decrease. Crosslinking did not favor enzymatic activity for the support under study, as the strengthening of interactions between the enzymes and the mycelium, combined with  the support’s porosity, hindered access to the substrate immobilized within it. Therefore, the non-crosslinked biocatalyst is more suitable for FOS synthesis in batch process, as it  eliminates an additional processing step while maintaining high catalytic performance.