Enzymatic Synthesis Of Sucrose Coupling Products Separation Online In Two-Phase Systems

The sucrose-6-acylates is the key intermediates in the preparation of sucralose which is a commonly consumed high intensity sweetener. Sucralose is a new non-nutritive sweeteners, has broad applications in food industry, cosmetics, oral-care products and medical supplies. Sucrose esterification was investigated in aqueous-organic two-phase systems. Sucrose dissolved in aqueous phase, and this favored the synthesis of sucrose ester. Meanwhile, vinyl acetate dissolved in organic phase, it could avoid the excess hydrolysis of vinyl acetate.To easily recycle and reuse Candida rugosa lipase with excellent catalytic properties, resins were screened as the carriers to immobilize the Candida rugosa lipase by adsorption. The results showed that the lipase immobilized on anionic resin 214 had the highest specific activity. The optimal conditions of immobilization were as follows:buffer pH=8.4, adsorption time 3 h, concentration of Candida rugosa lipase 8 mg/mL and temperature 35 ℃. And immobilized enzyme continuously catalyzed transesterification of sucrose with vinyl acetate was investigated. Previous screening tests showed that the optimal two-phase system was determined to be Tris-HCl/tert-amyl alcohol. Several factors that affect sucrose acylation were investigated in detail, including molar ratio of vinyl acetate to sucrose, initial buffer pH, buffer content, sucrose quantity, reaction temperature and lipase quantity. The optimal conditions for synthesizing sucrose acetate were achieved as follows:molar ratio of vinyl acetate to sucrose 2:1, initial buffer pH 8.4, ratio of tert-amyl alcohol to buffer 5:5 (v/v), sucrose quantity 2 mmol, reaction temperature 45 ℃, and lipase quantity 0.3g. Under these optimized conditions, the yield of sucrose acetate reached up to 1.4728 g, the selectivity of 6-sucrose acetate reached 50.37%. After replenishing tert-amyl alcohol, the activity of immobilized enzyme kept high after 10 cycles of reactions in this system.The study focused on optimization of catalyzed sucrose acylation in a two-phase system, which consisted of buffer solution and different organic solvents. The acetylation yield of non-enzymatic reaction was similar to lipase-catalyzed reaction in Tris-HCl/sec-butyl alcohol two-phase systems. The optimal conditions for synthesizing sucrose acetate were achieved as follows:molar ratio of vinyl acetate to sucrose 2:1, pH 9.0, ratio of sec-amyl alcohol to water 4:6 (v/v), sucrose concentration 600 mmol/L, and reaction temperature 45 ℃. Under these optimized conditions, the yield of sucrose acetate reached up to 48.29%, the selectivity of 6- sucrose acetate reached 45.51%. After enlarging the volume of reaction to 100 mL, the yield of sucrose acetate approximately reached 40.23%, the selectivity of 6-sucrose acetate reached 42.02%.