| Vitamin A and its derivatives are widely applied in the fields of comestics,health dink and dermatological therapy,and possess many healing efficacies such as keeping the retina normal,anti-inflammatory,resisting oxidation and regulating immunity.Vitamin A derivatives are more stable than vitamin A,especially vitamin A palmitate which is most widely used among them.Currently,vitamin A derivatives are mainly produced by chemosynthesis at home and abroad,which is energy-consuming,polluting and could form isomeride.Therefore,it is important to establish a green manufacturing route for lipase-catalyzed synthesis of vitamin A derivatives with mild reaction conditions and high selectivity.In this thesis,textile is used as carrier to immobilize lipase by adsorption and covalent binding,respectively.The optimal conditions for adsorbent immobilization were determined as follows:35℃,cotton-1 was used as the carrier,the proportion of textile and activating solution was 1:7,the concentration of lipase in the immobilization system was 19.75 g/L,the time for immobilization was 5 h,and the specific activity of immobilizaed lipase was 750 μmol/L/min/g;the optimum conditions for covalent immobilization were following:cotton-1 was used as the carrier,the concentration of NaIO4 was 0.3 mol/L,the buffer of citric acid-sodium citrate whose pH value was 5.0 was used as immobilization system with a ionic strength of 10 mmol/L,the concentration of lipase was 2.08 g/L,the time for immobilization was 1 h,and the specific activity of immobilizaed lipase was 915 μmol/L/min/g.The catalyzing synthesis of vitamin A palmitate with lipase immobilized by conalent binding,and the optimal conditions for transesterification were determined as follows:30℃,180 rpm,n-henaxe was adopted as reaction system,the concentrations of vitamin A acetate and palmitic acid were 10 g/L and 46.8 g/L respectively,the dosage of immobilized lipase was 30 g/L,catalyzed for 6 h,and 807%of conversion was yield;the substrate spectrum of the lipase to fatty acids was investigated when using vitamin A acetate as one of substrate,showing that the lipase have high catalyzing activity to long-chain fatty acids,as well as short-chain fatty acids.Finally,the kinetics of the lipase in the synthesis of vitamin A palmitate was studied,indicating that the reaction was corresponded with ping-pong Bi Bi mechanism,the values of Vmax,KmA,KmB were 72.464 μmol-min/g,0.210 mol/L,0.202 mol/L,respectively.The purification and analysis methods for lipase-catalyzed synthesis of vitamin A palmitate using vitamin A acetate as substrate were studied.The quantitative processing conditions for extraction of vitamin A palmitate were identified:the extraction system is ethanol-water and n-hexane,the concentration of ethanol is 80%,the ratio of volumes of ethanol-water and n-hexane is 3:1,the temperature and cycle for extraction are-20℃ and 7,respectively.The ultimate mass fraction of vitamin A palmitate reached 96.4%.After extraction,the silica gel chromatography was applied to further separate and purify vitarmin A palmitate:1 mL of the reaction mixture was loaded to silica gel column(200～300 mesh,20×280 mm)and ethyl acetate-petroleum ether(1:9,V/V)was used as mobile phase;The flow rate is 68.5 mL/h and the eluent is collected per 5 mL.The result showed that vitamin A acetate and vitamin A palmitate can be completely separated by silica gel chromatography. |
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