Enzymatic Synthesis And Technological Design Of Vitamin A Palmitate

Vitamin A palmitate is widely used in cosmetics,nutrition and health care,as well as skin therapy drugs,whose physiological function is identical to vitamin A but more stable than vitamin A.Candida antarctica lipase B(CALB)is a high-efficiency lipase which catalyzes hydrolysis,esterification,transesterification of broad spectrum of substrate,widely applied in the non-aqueous phase catalysis.Currently,the vitamin A derivatives are mainly produced by chemical synthesis,which will consumpt high-energy,pollute heavily and easily produce isomers.Meanwhile,the enzymatic synthesis of vitamin A derivatives has made great progress in recent years.This study investigated the efficiency of fermentation and optimized the enzyme producing conditions of Pichia pastoris by using the single factor and the orthogonal experiment methods.The optimum in 700 L fermenter with a liquid volume of 400 L was determined:pH 5.0,30?,250 rpm,5%of inoculum,20-30%of dissolve oxygen,129 L methanol as a inducer flowing in,160-170 h for culture.And our results discovered that addition of 0.5%yeast extract and 1%peptone(the volume ratio of medium)to the fermentation broth after inducing further improved the fermentation broth volume activity with a fed-batch every 48 h,a total of three times.Finally,the enzyme activity reached 18 U/ml.Processing of lipase-catalyzed synthesis of vitamin A palmitate has two steps.The first step is the hydrolysis of vitamin A acetate to vitamin A alcohol and the second is to use enzyme to synthesize product by esterification.Our data determined the optimal conditions for the hydrolysis:concentration of KOH is 375 g/L,substrate concentration is 100 g/L,hydrolysis temperature is 20?,hydrolysis time is 50-60 min.The optimal esterification conditions:molar ratio of vitamin A acetate and the palmitic is 1:4,immobilized enzyme is 30 g/L,the reaction temperature is 40?,anhydrous Na2SO4 is 125 g/L(or MgS04 50 g/L).The separation and extraction of lipase-catalyzed esterification were studied to determine the key factors which affected the efficacy.The results revealed that palmitic acid crystallized in 4? with 18-24 h was removed by filtration,and then the crude product was obtained.Optimal distillation temperature was 35?,and when the concentrate was diluted 2 times with hexane in 4? recrystallization remove palmitic acid again.In the process of extraction and separation,the condition for producing 99%pure vitamin A palmitate was 95%methanol-water aqueous and crude product with the ratio of 1:3,extraction temperature of 30?,and 4 times with 20 mins' extraction and 10 mins' delamination.Ultimately,the study designed three parts of producing vitamin A by the application of biological method,namely fermentation,biocatalysis and extraction and estimated the technological design from material balance and equipment briefly.Also,the results will provide a basis for the scale of 200 t/a of vitamin A palmitate.