Synthesis Of Galactooligosaccharides By β-Galactosidase And Its Immobilization

Galactooligosaccharides(GOS) are low-caloric oligosaccharides consisted of 2~10 galactose units and one terminal glucose residue. GOS are recognized as prebiotics that stimulate the growth and the activation of desired bacteria in the colon. Conresspondingly, they promote mineral absorption, reduce serum cholesterol level and prevent tooth decay. These physiological effects, together with physicochemical characteristics, have enabled GOS be widely used in a variety of design foods. Commercially, GOS are produced from lactose by enzymatic synthesis using β-galactosidase, under kinetic control caused by the competition between the reactions of hydrolysis and transgalactosylation. However, free β-galactosidase was not able to separate from the reaction system and wasted. From an economical and technical point of view, the immobilization of the biocatalyst may convey significant improvements in any enzymatic process, which could recycle enzyme and thus save production cost. The main contents in this study are as follows:Firstly, Bacillus circulans SK28.003 was cultured in medium fermentation tank(30L) for 80 h, with β-galactosidase activity reaching 2.48 U/m L in the enzyme solution. β-galactosidase was concentrated by ultrafiltration. The specific activity was increased from 720.0 U/g to 1240.0 U/g, and the recovery was 63.4%. 50.5 g wet β-galactosidase protein were obtained by concentration, precipitation and centrifugation. 7.9 g enzyme powders(3195.3 U/g) were obtained by freeze drying, with enzyme activity recovery of 56.5%. After storage for 12 months in the fridge(-20 °C), β-galactosidase powder still remained 80% of original activity, indicating good storage stability.Secondly, to increase the efficiency of transgalatosylation and GOS yields, the optimal reaction conditions, such as initial lactose concentration, loading amount of the enzyme, temperature and p H of the buffer, were determined through single factor and orthogonal experiments. When the GOS synthesis was carried out at 60 °C using 500 g/L lactose and 6 U/g lactose in 0.1 M sodium phosphate buffer at p H 7.5, the maximum GOS yields were achieved by 45.5% after 12 h.Thirdly, β-galactosidase was immobilized on DEAE cellulose through crossing-linking reaction. The optimal conditions for the immobilization were determined as following: 1.0 g DEAE cellulose was pretreated with 1.0% solution of glutaraldehyde, then 9.0 U β-galactosidase was immobilized on the carrier, with temperature 8 °C, p H 6.5, adsorption time 7 h. Under these conditions, the activity of the immobilized enzyme reached 2.0 U/g and the recovery of enzyme protein was 78%. The characteristics of the immobilized enzyme were obtained. The optimum reaction temperature was 50 °C, which was consistent with the free enzyme. The optimal reaction p H 5.5 was shifting to acidity. Thermal, p H and storage stabilities of β-galactosidase were improved after it was immobilized on DEAE cellulose. The immobilized enzyme reaction column showed good operation stability as the conversion rate of lactose still reached 70% after 5 batches.Finally, Fe3O4 nanoparticles were synthesized by chemical co-precipitation with amino groups coated on the surface. These magnetic nanomaterials were characterized by XRD, FTIR and TEM technologies. The results indicated that magnetic Fe3O4 structure was successfully carried out with 1,6-hexanediamine as amine-functionalization reagent, which was suitable for immobilization and simultaneously improved the dispersibility in solutions. Based on their reactive functional group, excellent biocompatibility and magnetism, the magnetic Fe3O4 nanoparticles were applied for β-galactosidase immobilization. The optimum conditions for immobilization were as follows: 36 U of enzyme per gram support, 25 °C, p H 7.5 for 6 h. The properties of immobilized β-galactosidase were studied. The optimal p H and temperature for reaction was 7.5 and 50 °C respectively. After 6 batches, approximately 42% of the biocatalyst initial activity was retained, indicating good operation stability.