The Chemo-enzymatic Synthesis Of Vitamin A Palmitate

Vitamin A is a kind of natural and fat-soluble nutrient substance,which is essential and involved in a variety of physiological processes of body.Because of the unstable characteristic of vitamin A,commercially available vitamin A is in the form of vitamin A ester derivatives mainly including vitamin A acetate and vitamin A palmitate.Vitamin A palmitate is more stable and not easy to be damaged and the usage tends to be widespread in cosmetics,medicine,feed etc.At present,the production of vitamin A palmitate still primary relies on chemical synthesis.It is urgent to seek a kind of green manufacturing process in the light of the shortcomings of chemical method.The study was launched in three aspects containing the immobilization of free lipase,the alkaline hydrolysis of vitamin A acetate and the esterification synthesis of vitamin A palmitate by immobilized lipase in non-aqueous system.In order to obtain relatively pure lipase powders used in the immobilization,high-density fermentation of Pichia pastoris X-33/CALB established in our laboratory was performed.The curves of cell growth and lipase production in fermentor were determined.The wet cell weight reached 311.0 g/L and the enzyme activity reached 8.0 U/mL.After purification,the final purification fold was 1.8 times and the yield of lipase was 81.6%with specific activity 8.1 U/mg.Macroporous adsorption resins were used as the carrier and free CALB was immobilized by physical adsorption method.The immobilized conditions were optimized as follows:the macroporous adsorption resin AB-8 as the immobilized carrier;disodium hydrogen phosphate-citric acid buffer(ionic strength controlled with Na2HP04 to 0.05 M and citric acid to 0.025 M)pH 6.0;lipase concentration of 16 g/L.The free lipase was adsorbed on AB-8 with the ratio of support to enzyme solution of 1:20(w/v),incubating at 35? and 180 rpm for 2 h in a water bath.The adsorption ratio of enzyme protein was 87.5%;the specific activity was 1034.4 U/g immobilized lipase.Simultaneously,the enzymatic properties of the immobilized lipase were studied.The hydrolysis of vitamin A acetate in alkaline conditions was investigated.In the hydrolysis process,co-solvent was added to improve the rate of hydrolysis because vitamin A acetate is insoluble in water and not be directly hydrolyzed with alkali liquor.The optimum hydrolysis conditions was 10 g of vitamin A acetate,8 mL anhydrous ethanol,10 mL 5 M KOH solution at 16?,200 rpm for 1 h.The hydrolysis ratio of 100%was obtained.Immobilized lipase prepared was applied in biocatalytic synthesis of vitamin A palmitate and the conditions of esterification were further studied.A certain amount of ethanol could dissolve in non-aqueous solvents in the process of extraction.Immobilized enzyme not only catalyzed the synthesis of vitamin A palmitate but also the synthesis of ethyl palmitate in the esterification reaction,which made the separation process of product more complicate and difficult and greatly affected the yield of vitamin A palmitate at low substrate molar ratio conditions.It was a necessary process to remove ethanol in esterification reaction system in case the formation of ethyl palmitate.The parameters of esterification reaction were predicted as follows:non-aqueous solvent(n-hexane),extracted liquid washing six times,substrate concentration(350 g of vitamin A acetate per liter),substrate molar ratio of vitamin A alcohol to palmitate(1:1.1),immobilized enzyme concentration(30%w/w of vitamin A acetate),0.5 g anhydrous magnesium sulfate,temperature(30°C),and reaction time(1 h)at 180 rpm.Under these circumstances,recombinant lipase-catalyzed esterification of vitamin A alcohol could reach a yield 96%.Liquid-liquid extraction was proven to be a simple and effective method of the separation of vitamin A palmitate.The yield could be reach 80.3%with final purity 99.0%.