Study Of Lipase-Catalyzed Synthesis Of L-Asborly Fatty Acid Esters

L-ascorbyl fatty acid esters can be used widely as natural additives in food, medicine and cosmetics with fat-soluble antioxidant and emulsifier characteristics. Relative to the chemical synthesis, the enzymatic synthesis could be performed under milder conditions and the product obtained with higher security, so it can be considered to be a synthetic route with more environmentally friendly. However, economy substrate, the stability of enzyme and production engineering are limiting factors for the development of enzymatic synthesis. In this study, lipase-catalyzed synthesis of L-ascorbic acid fatty acid esters was investigated deeply with palm oil as acyl donor, and the exploration of continuous reaction process was in favor of the industrialization of enzymatic synthesis technology.Firstly, the enzymatic synthesis reaction system was investigated to determine the organic phase as the reaction medium. Though the study of various factors to the enzymatic reaction, the result revealed the solvent type and water activity (aw) of system are key factors. By removing the water of solvent with molecular sieves sufficiently and adding the molecular sieves to the system at the beginning of the reaction can keep the low aw effectively, thereby increasing production. The optimal reaction condition in tert-amyl alcohol system was determined:With 0.352 g ascorbic acid and 4.535 g palm oil catalyzed by 14.2% Novo435 (weight % of ascorbic acid) in 20 mL tert-amyl alcohol,50 g/L molecular sieve was added, rotation speed 200 r/min, the product concentration is 22 g/L after 24 h reaction at 55℃. The significant factors affecting the reaction were optimized by response surface experiment and the optimum condition was obtained:with 90.32 mg Novo435 and molar ratio 9.11, at 55.2℃, the maximum theoretical conversion is 74.31%.Then, the fed-batch reaction process of enzymatic reaction was studied at the most optimal conditions. The adding time, additional amount and times were determined, this process could make substrate and enzyme utilize effectively and improve the yield per unit volume. The effect of enzyme pretreatmenition on the catalytic efficiency and stability of enzyme was explored and studied, the result showed that initial reaction rate could be improved significantly when the enzyme pretreated by soaking with tert-butyl alcohol and palm oil. Subsequently, combined with the less catalytic stability of enzyme during the process of repeated using, a method for enzyme regeneration was designed:the enzyme was regenerated by soaking with tert-butyl alcohol and substrate when repeated using, the relative activity of enzyme could still maintain 85% and increase 1.07-fold to the group with not regenerated. This method can improve the enzymatic stability and reduce the catalyst cost.Finally, the route for continuous enzymatic synthesis process of L-ascorbyl fatty acid ester was designed. In this process, the product and residual palm oil was separated by extracting with ethanol (95%), thus break the limitation of equilibrium which induced by the product accumulation. Then a certain amount of substrate added, tert-amyl alcohol and the regenerated enzyme were used to continuous catalyzed reaction, the total conversion remained at 65% and production increased 1.36-fold compared with fed-batch process, besides the product concentration remained stable. The continuous synthesis process make the substrate, enzyme and solvent use continuously, reduce the production coat and improve the production efficiency significantly, so it have a certain significance to the industrial application of enzymatic synthesis.