Production Of Lactulose With Immobilized Lactase On Magnetic Chitosan Microspheres

Enzymatic methods for lactulose synthesis are novel approaches for lactulose preparation. Immobilization of lactase on magnetic chitosan microspheres and production of lactulose with immobilized lactase on magnetic chitosan microspheres were two parts investigated in this thesis. The preparation of microspheres, the optimization of immobilization process, the properties of the immobilized lactase and synthesis of lactulose in a two-phase system were discussed, respectively.Fe3O4 was synthesized by co-precipitation. When n(Fe²⁺):n(Fe³⁺) was 1:1.75, reactive temperature was 45℃, ripening temperature was 70℃, pH was 10, reaction time was 1.5 h, the magnetic fluid with high magnetism and small diameter can be obtained. Magnetic chitosan microspheres were prepared via suspension cross-linking technique in the application of the formaldehyde. When Span-80 was 5⁸%(v/v), chitosan was 1.5%(w/v), the ratio of chitosan/Fe3O4 was 3:4, formaldehyde was 5%(v/v), and the stirring rate was 1000 r/min, the magnetic chitosan microspheres had good enzyme binding capacity and can be used as good carriers to immobilize lactases.The immobilization of lactase onto magnetic chitosan microspheres by cross-linking with glutaraldehyde was investigated. It was found that magnetic chitosan microspheres should swell in buffer (pH7.0) buffer for 12 h. The optimum immobilization conditions for lactase were: 1.6 U/mg lactase linked with magnetic chitosan microspheres for 30 min and then reacted with 0.2%(v/v) glutaraldehyde at the same temperature for 5 h. The activity of immobilized lactase was 420 U/g, and the activity recovery was 26%.Moreover, the optimal temperature of immobilized lactase was 35℃, which was 5℃higher than that of free lactase. At the same time, the optimal pH of immobilzed lactase was 8.0, which was 1.5 higher than that of free lactase. Nevertheless, immobilized lactase exhibited better thermal stability and pH stability. The Km value was 73.3 mmol/L when O-nitrophenylβ-D-galactopyranoside was used as substrate, which was higher than that of the free lactase (12.95 mmol/L). This indicated that the affinity between enzyme and substrate decreased significantly.The preparation of lactulose by lactase was carried out in cyclohexane /water two-phase system. The optimal preparation conditions of lactulose by immobilized lactase and immobilized glucose isomerase were studied. And the optimum conditions were obtained as following. In 20 mL cyclohexane/water (95:5,v/v) two-phase system, lactose content was 0.8 g, fructose content was 0.1 g, magnetic immobilized lactase was 0.1 g, and IGI was 0.05 g, the reaction temperature was 30℃, pH was 8.0, the reaction time was 2 h. The final yield of lactulose production was 151 g/L, which was three times higher than that in aqueous system.