Marija Ćorović

Novel β-galactosidase nanobiocatalyst systems for application in the synthesis of bioactive galactosides

In this study, unmodified, amino modified and cyanuric chloride functionalized amino modified nonporous fumed nano-silica particles (FNS, AFNS and CCAFNS, respectively) were used for the development of efficient nanobiocatalysts for application in the biosynthesis of bioactive galactosides, galacto-oligosaccharides (GOS). Hence, in an attempt to elucidate the mechanism of immobilization, based on available enzyme conformation, the effects of immobilization parameters (initial enzyme concentration, immobilization time and pH) were analyzed. Among all three used nano-sized supports, the one with amino groups (AFNS) exhibited the best β-galactosidase binding capacity of 220 mg g−1 support with the efficiency of ∼90% at pH 4.5 and immobilization time 3 h. The highest hydrolytic activity of ∼2200 IU g−1 was achieved, which is far higher than previously reported. Meanwhile, β-galactosidases covalently immobilized on CCAFNS and adsorbed on FNS nanoparticles were found to have similar trends with respect to immobilization efficiency (58–71%) and hydrolytic activity (∼600 IU g−1). Furthermore, thermal stability at 60 °C was increased by β-galactosidases immobilized on AFNS and CCAFNS (4 and 1.4-fold, respectively) due to the protective effect of interactions formed between enzyme molecules and the surfaces of nanoparticles. Since β-galactosidase immobilized on AFNS was the nanobiocatalyst with the highest activity and stability, it was applied in GOS production. With AFNS–β-galactosidase GOS production of 90 g L−1 h−1 was achieved as compared to 30 g L−1 h−1 by free β-galactosidase, meaning that AFNS enhanced the selectivity of β-galactosidase for transgalactosylation, which is a crucial advantage for its application in GOS production.

Optimization of galacto-oligosacharides synthesis using response surface methodology

Galacto-oligosaccharides (GOS) are important lactose-derived compounds, considered to be a prebiotics, based on abundant scientific evidence about their unique physical properties and physiological effects. This consequently allows their widespread application as supplement in food and feed industry. They are preferably produced by the enzymatic transgalactosylation action of βgalactosidase. However, this enzyme simultaneously performs its primary biological function of lactose hydrolysis, and it is of crucial importance to gain an insight into the influence of different reaction conditions, and provide favorization of transgalactosylation, particularly GOS synthesis reaction. In this study, the response surface methodology (RSM) was applied in terms of individual experimental factors effect estimation, their mutual interaction identification and finally, the determination of optimum conditions for highest GOS yield achievement. Having said that, it can be observed that the temperature and pH have no significant impact on the GOS yield, while on the other hand, the lactose concentration of 400 g/l, enzyme concentration of 13.5 g/l and reaction time of 13 min represent the optimum conditions for achieving the highest GOS yields.