Imobilização de frutosiltransferase microbiana em gel de alginato e sua caracterização para a produção de frutooligossacarídeos

Abstract

Fructooligosaccharides (FOS) are sucrose molecules containing fructosyl units linked by β (2→1) position. These prebiotics are low calorie sweeteners widely used in formulations in food and pharmaceutical industry. Obtaining FOS via enzymatic route with sucrose as substrate and enzymes of microbial origin called fructosyltransferases (FTases), has been studied by IPT (São Paulo), using strains of the fungus Aspergillus oryzae to obtain the mycelial extract. Researches in progress at the Bioprocess Laboratory (UNIFAL), along with IPT, use these extracts to evaluate and optimize the FOS production. The present proposal aims to complement these investigations by the immobilization of the mycelial extract previously crosslinked with glutaraldehyde by entrapment in calcium or barium alginate beads. Initially, beads production conditions, like formulations, level of mechanical dispersion of the mycelium, type of alginate degree of crosslinking and characteristics of the material (diameter and aspect of the beads, mechanical strength, stability at storage) were evaluated. The optimization of the entrapment conditions was based on enzymatic activity under standard reaction conditions and was complemented by conducting the batch transfructosylation reaction under different temperatures, pHs, substrate concentrations and reuse of the spheres. The immobilization condition that produces the highest FOS yields used the low viscosity alginate (4.0% w/w) crosslinked with 0.2 mol.L-1 calcium chloride and the smallest diameter beads. These optima conditions generated spherical beads, with homogeneous, porous and continuous surfaces, with larger resistance to mechanical compression and higher storage time stability at 4 °C. The temperature of 50 °C, pH of 5.5 and substrate concentration of 400 g.L-1 provided the maximum transfructosylation activities (ATs) and selectivity. The AT values ​​found with the glutaraldehyde crosslinked biocatalyst, disintegrated and then immobilized were about 70% larger than with the free, not dispersed biomass. pH stability tests showed that free biomass was stable in a pH range between 4.5 and 6.5, and the immobilized biocatalyst in the range of 5.5 to 7.5. The thermal stability analysis indicated that the immobilization of the biomass provided an increase in thermostability. Application of the biocatalyst in operational stability tests confirmed that immobilized biomass remained stable after six cycles of FOS production showing only 4% attenuation to initial activity, while the degradation rate of the free biocatalyst activity was about 10% per cycle. In cycles with total sucrose depletion (4 hours each), the immobilized biomass retained 92% of its initial activity until the third cycle, while using the free biomass resulted in a constant drop of about 15% in AT per cycle. Regarding the kinetics of the enzymatic reaction, the Hill model fitted more satisfactorily to the data than the Michaelis-Menten (MM) model, with a reduction in the Km (MM) and K0.5 (Hill) values and an increase in the maximum reaction rate with immobilized biocatalyst in relation to the free biomass. The bulk of these expressive gains evidences the potential of the application of Aspergillus oryzae IPT-301 mycelial FTase in FOS production after crosslinking the mycelium with glutaraldehyde and its involvement in calcium alginate beads.