Dual-enzymatic synthesis of lactulose in organic-aqueous two-phase media

Lactulose has been successfully synthesized by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials. Cyclohexane–buffer system C6H12:buffer = 95:5 (v/v) was employed as the organic-aqueous media for the reaction. The dual-enzymatic system was consisted of immobilized lactase (IL) and immobilized glucose isomerase (IGI). Immobilized lactase was prepared by cross-linking the free lactase into Fe3O4-chitosan magnetic microspheres. The main enzymatic reaction parameters were investigated, including reaction temperature (T), pH value and reaction time (t). Under the optimum reaction conditions, i.e., lactose 0.8 g mL−1, fructose 0.1 g mL−1, IL 0.1 g mL−1, IGI 0.05 g mL−1, T = 30 °C, pH = 8.0 and t = 2 h, the obtained highest lactulose yield was approximately 151 g L−1 and the corresponding productivity was 75.5 g L−1 h−1. Experimental results indicated that the organic-aqueous media can significantly promoted the transglycosidation activity of lactase and therefore improve the lactulose yield. The possible reaction mechanism of the synthesis of lactulose using IL and IGI in two-phase system was also proposed.