The Production And Immobilization Of Candida Antarctica Lipase B And Its Application In Preparation Of Vitamin A Palmitate

Vitamin A palmitate is currently one of the main ester derivatives of unstable vitamin A alternatives on the market.Vitamin A palmitate is compare to vitamin A more stable and not easy to be damaged and is widely used in medicine,cosmetics,feed et al.At present,the production of vitamin A palmitate on market primary relies on mature chemical synthesis.It is urgent to seek a kind of green manufacturing process considering the environmental protection etc.This study selected several immobilized carriers,optimized the lipase immobilized method,and applied it on novel chem-enzymatic synthesis of vitamin A palmitate.The study was launched in three aspects containing the obtain of Candida Antarctica lipase B,the immobilization of free lipase and the esterification synthesis of vitamin A palmitate by immobilized lipase in non-aqueous system.In order to obtain relatively pure lipase powders,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 326.9 g/L and the enzyme activity reached 8.3 U/mL.After purification,the final purification fold was 3.0 times and the yield of lipase was 77.7%with specific activity 7.8 U/mg.Three kinds of resins ECR1030M?LX-1000EP and MC150EP in 10were selected for the better immobilized effect.The immobilized conditions by physical adsorption method were optimized as follows:Disodium hydrogen phosphate-citric acid buffer p H8.0?immobilized lipase by ECR1030M and MC150EP?or pH6.0?immobilized lipase by LX-1000EP?;18 g/L lipase solution and resin were mixed with 20:1?v/w?,incubating at 30°C and 180 rpm for 3 h in a water bath.The pecific activity was 1055.7 U/g immobilized lipase by ECR1030M,1077.0 U/g immobilized lipase by LX-1000EP and 1024.3 U/g immobilized lipase by MC150EP.Simultaneously,the enzymatic properties of the immobilized lipase were studied.Two kinds of immobilized lipase by resins ECR1030M and LX-1000EP were applied in biocatalytic synthesis of vitamin A palmitate and the conditions of esterification were further studied.In this study,anhydrous ethanol was selected as co-solvent in chemical hydrolysis to contribute the solubility of vitamin A acetate and further to improve the hydrolysis rate of the vitamin A acetate.However,immobilized lipase also could simultaneously catalyze palmitic acid with ethanol to form ethyl palmitate during the esterification to prepare vitamin A palmitate,which makes the esterification process require too much palmatic acid and the downstream separation process of product more complicated and difficult.So to avoid the formation of ethyl palmitate,the ethanol was removed by distilled water before esterification reaction.The parameters of esterification reaction were predicted as follows:non-aqueous solvent?n-hexane?,extracted liquid washing 4 times,substrate concentration?300g of vitamin A acetate per liter for immobilized lipase by ECR1030M and250 g of vitamin A acetate per liter for immobilized lipase by LX-1000EP?,substrate molar ratio of vitamin A to palmitate?1:1.1?,immobilized enzyme?25 g/L?,anhydrous magnesium sulfate?50 g/L?,temperature?35°C?,and reaction time?1 h?at 180 rpm.Under these circumstances,The yield of vitamin A palmitate could reach 97.9%and96.8%.The pure vitamin A palmitate?99%?was separated and purified by the two-step freezing crystallization method.The structure identification of the pure vitamin A palmitate were performed with infrared spectrum,mass spectrum,¹H NMR and ¹³C NMR.Compared with the standard,the reaction products was indeed the compound of vitamin A palmitate.