The Synthesis Of L-Ascorbyl Palmitate Catalyzed By Immobilized Lipase In Non-Aqueous Medium

L-Ascorbyl Palmitate (L-AP) is a kind of fat-soluble antioxidant, and has good radical scavenging, antioxidant, anti-aging, and anticancer functions. L-AP has been widely used as an additive in foods, oils, cosmetics, and etc. In present market, commercial L-AP is basically synthesized by chemial methods in the presence of concentrated sulfuric acid. However, chemical synthesis has many disadvantages, such as discharge of acid wastewater, equipment erosion by concentrated sulfuric acid, by-products, and etc.Enzymatic catalysis has been developed for its mild reaction conditions, high selectivity, high catalysis efficiency, and environmental friendliness. Furthermore, enzyme immobilization overcomes the shortcomings of free enzyme, such as less stability, easy deactivation, high cost, and low reusability. This makes enzyme-catalysis has significant predominance relative to chemial synthesis, particular in non-aqueous medium.In this study, immobilized Candida sp.1619 lipase was used to synthesize L-AP. The parameters affecting the synthesis of L-AP, and the effect of product inhibition and by-product on synthesis of L-AP were investigated. Also, the feasibility of continuous synthesis of L-AP catalyzed by immobilized lipase was studied. The main results as followed:(1) Lipase (Candida sp.1619), immobilized on hydrophobic PTFE membrane with nonwoven fabric by a procedure combining filtration, adsorption and protein cross-linking, has good storage stability and operational stability. Residual activity of immobilized lipase remained 65% of its initial activity after 380d storage in natural environment, and its halflife was 140-160h.(2) The influences of solvent, temperature, time, substrate molar ratio and concentration, dosage of immobilized lipase, and water activity on the synthesis of L-AP were investigated. The results showed that tert-amyl alcohol was a favorable solvent for the biosynthesis of L-AP in present study. Other optimal conditions were:reaction temperature,55℃; reaction time,24h; substrate molar ratio,9:1; substrate concentration,0.79mol-L-1; dosage of immobilized enzyme,100U/20mL solvent; water activity,0.1-0.3. Under above conditions, L-AP concentration reached 14g·L-1.(3) Effects of product (L-AP) and by-product (water) on L-AP sythesis were inverstigated. The results showed that both L-AP and water had significantly negative impact on further synthesis of L-AP. However, L-AP synthesis can benefit from water removal from reaction system by adding adequate zeolite molecular sieve at right time. L-AP concentration reached to 25.9g-L-1 when 0.6g zeolite molecular sieve (4A) was added to the reaction system after 8h of experimental beginning, which was higher by 80% than that of without molecular sieve.(4) The feasibility of continuous synthesis of L-AP catalyzed by immobilized lipase was studied. The results showed that L-AP sythesis was better at flow rate of 45mL-min-1 and retention time of 2min. Adding molecular sieve to the reaction system greatly facilitated the continuous synthesis of L-AP, and its concentration increased by about 70% after 40h of reaction compared to that of without zeolite molecular sieve.