β-Galactosidase is the industrially important enzyme that catalyzes both, lactose hydrolysis and synthesis of different bioactive galactosides. In this study, optimal conditions (fermentation temperature, inoculum concentration and lactose concentration) for accomplishing high yields of β-galactosidase activity from Lactobacillus acidophilus ATCC 4356, bacteria regarded as safe for human consumption, were investigated. Using the response surface methodology (RSM), it was concluded that the highest activity and specific activity were obtained by 2-day shake-flask culture fermentation at 28 °C, provided that the lactose content in the fermentation medium was 1.48%, and the inoculum concentration was 2.8%. The optimum temperature and pH for the obtained enzyme were 45 °C and 6.5, respectively. More importantly, these conditions simultaneously ensure a high enzyme stability. The Km and Vmax values were 0.44 mM and 25.64 mM/h (for o-nitrophenyl-β-D-galactopyranoside), and 3.79 mM and 3.10 mM/h (for lactose), and the substrate excess inhibition was not observed. On the other hand, the enzyme was inactivated in the presence of Ca 2+ , Ba 2+ , and Cu 2+ .

In this study, immobilization of laccase from Trametes versicolor on eight Lifetech? supports, with different characteristics (pore size, length of the spacer arm and functional groups), was studied and optimized for intended use in bioremediation for decolorization of industrial wastewaters. Out of six tested amino-functionalized supports, the most promising carrier was proved to be porous Lifetech? ECR8309F with primary amino groups and a C2 spacer arm. Onto this support, laccase is attached by forming electrostatic interactions so that the most active preparation has shown the activity of 66876 U/g support. On the other hand, during immobilization of laccase on epoxy-functionalized Lifetech? ECR8285F, via hydrophobic interactions and covalent bonding confirmed by a desorption assay, immobilization yield of 60 % and the activity of 118929 U/g were accomplished. Furthermore, immobilized enzyme on this support showed high capacity for decolorization of dyes (Lanaset? Violet B, Lanaset? Blue 2R, bromothymol blue and bromocresol green), by combination of both adsorption and enzyme degradation. Decolorization was in the range of 88 to 96 % after 4 h, with more than 80 % achieved after only 45 min. Also, this preparation demonstrated high operational stability during seven consecutive reuses in all examined dye reaction systems.[Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. 451-03-68/2020-14/200135 and Grant no. 451-03-68/2020-14/ 200287]

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 β-galactosidase. 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.

Lipophilic derivatives of vitamin C are additives with antioxidant properties, attractive for application in food, cosmetics and pharmaceutics. They could be synthesized in lipase-catalyzed processes by using various acyl donors. Hereby, we present application of linoleic acid, which is polyunsaturated fatty acid essential in human nutrition, for esterification of vitamin C catalyzed by immobilized enzyme preparation Novozym ® 435 in acetone. Highest specific ester yield, 9.7 mmol/g of immobilized lipase, was accomplished with 0.15 M of vitamin C, 0.6 M of linoleic acid, 3 g/l of enzyme and 0.07% (v/v) of water, at 60 °C. NMR analyses of purified product proved that synthesized molecule was identical to 6-O-ascorbyl linoleate. Capacity of ester for scavenging 2,2-diphenyl-1-picrylhydrazyl radicals was two times higher comparing to parent molecule, vitamin C. Its diffusion coefficient, determined using Franz cell and cellulose acetate membrane, was 40% higher than palmitate and 62% higher than oleate. Obtained results showed that L-ascorbyl linoleate could be successfully synthesized in biocatalyzed processes. Furthermore, it was demonstrated that it possess high potential for application in different lipophilic products due to its liposolubility, high antioxidant efficiency and good diffusion properties.

Cellulases are enzymes which catalyse cellulose hydrolysis and are widely used in various industry branches. Lately, their application in treatment of different agroindustrial waste materials which could serve for fuel production is being extensively explored. In order to increase their stability and cost-effectiveness of their usage, application of their immobilized forms are preferred over free enzymes. Hereby, we tested eight different Lifetech TM immobilization supports differing in polarity, porosity and functional groups as carriers for Asspergillus niger cellulase immobilization. Most promising carrier was methacrylate based, with primary amino groups, C6 "space arm" and pores with diameter of 60-120 nm-Lifetech TM ECR8409F. For this support, most important immobilization parameters were investigated and after 3 h at pH 6 with initial protein concentration of 23.3 mg/g support immobilized cellulase with 406 IU/g (with carboxymethyl cellulose as a substrate) was obtained. This preparation was successfully applied in the hydrolysis of lignocellulosic fraction of sunflower seed meal, which is widely available byproduct of sunflower seed meal fractionation for protein-rich fractions production. Initial reaction rates and yields of reducing sugars were unchanged comparing to free enzyme, indicating that there were no significant diffusion limitations for substrate to approach active sites of A. niger cellulase molecules immobilized onto Lifetech TM ECR8409F support.