Synthesis Of Cinnamic Alcohol Ester Catalyzed By Candida Antarctica Lipase B-Displaying Pichia Pastoris Whole-cells

Cinnamyl alcohol esters are a kind of important aromatic compounds,Because of the pleasant fruit and floral scent,they are widely used in food,cosmetic and pharmaceutical industries.The synthesis of Cinnamyl alcohol esters is highly concerned by researchers at home and abroad.At present,most cinnamyl alcohol esters on the market are synthesized by chemical methods.There are many shortcomings in chemical synthesis methods,such as much of chemical catalysts,a variety of by-products,harsh reaction conditions,large energy consumption,difficult separation of products and so on.These shortcomings greatly affects the the quality of products.Nowadays,enzymatic synthesis of spices has attracted much attention because of its mild reaction conditions,high purity of product,good stereoselectivity and environmentally friendly.Therefore,finding a suitable biocatalyst for the enzymatic synthesis of cinnamyl ester and making the method can be used in a large scale has a wide range of economic benefits.Commercial lipase has a series of advantages,such as high reaction rate,easy to recycle and so on.But because of the high price,it can not be large-scale used in industrial.Depending on the characteristics that Pichia pastoris can efficiently express the foreign proteins and yeast cell surface display techniques,We let the exogenous lipase immobilized on Pichia pastoris cell surface.Furthermore we ferment it at a high density and obtain a large number of Pichia cell displaying CALB,after freeze-drying or spray-drying it can be used as a whole-cell biocatalyst in organic synthesis.The whole cell catalyst not only has the advantages of immobilized enzyme,but also easy to produce,which can effectively reduce the production cost.First of all,aiming at the cinnamyl ester used as flavor and fragrance in the market,we carried out a preliminary exploration for the synthesis of these ester by using the self-made lipase CALB displayed on the surface of Pichia pastoris(Candida antarctica lipase B-displaying Pichia pastoris,Pp-CALB)as catalyst.Then,the reaction conditions of Pp-CALB synthesis cinnamyl acetate was systematically explored and optimized.The synthesis of cinnamyl acetate by enzymatic transesterification was carried out in a solvent-free system,using vinyl acetate as an acyl donor via a whole cell biocatalyst Pp-CALB.The optimized reaction condition was: 6 mmol cinnamyl alcohol was added into a 10 ml Erlenmeyer flask,the initial water activity of the system was 0.53,the molar ratio of the substrate(vinyl acetate: Cinnamyl alcohol)2: 1,agitation speed 120 r/min,reaction temperature 50?,Pp-CALB 0.05 g.After 3 h reaction,the conversion reached 81.34%Recovered by filtration,washed and spray-dried,Pp-CALB was reused in the next reaction.The reaction conversion is still above 70.00% after reused 8 cycles.Pp-CALB showed good operational stability.On the basis of single factor optimization,taking the reaction temperature,molar ratio of the substrate,Pp-CALB dosage and agitation speed as the significant influencing factors,the four-factor and three-level orthogonal experimental was designed to study the influence of this 4 factors and their interaction on the conversion of cinnamyl alcohol.We analyzed the result and get the optimal reaction condition: reaction temperature 50?,agitation speed 120 r/min,Pp-CALB loading 0.06 g,substrate molar ratio(vinyl acetate: cinnamyl alcohol)1.5.In this condition,reaction conversion reached 89.00%,which was higher than the optimal conditions of single factor optimization(81.00%).On the basis of orthogonal experimental optimization,we use the 2L agitation reaction equipment to explore the large scale synthesis of cinnamyl acetate.Under the conditions of reaction volume 1L,initial water activity 0.53,Pp-CALB loading 31.3 g,substrate molar ratio 1.5,agitation speed 120 r/min,reaction temperature 50? and reaction time 3.5 h,the reaction conversion reached 88.00%,which is equal to the shake flask reaction level.The results showed that the whole-cell catalyst Pp-CALB could efficiently catalyze the synthesis of cinnamyl acetate in the reaction amplification,and its reproducibility is well.We also tried to purify and separate the cinnamyl acetate.The cinnamyl acetate product can be isolated by vacuum distillation and its purity reached 87.24%.This study provides the idea about using Pichia pastoris surface display lipase CALB to produce Cinnamyl acetate in industrial.