The Application Of Lipase In Cinnamyl Acetate Synthesis

In this thesis，application of lipase to the synthesis of esters is the major purposeof research. Comparing with different reaction routes or reaction systems, thesynthesis of cinnamyl acetate by lipase （Novozym435） through transesterificationroute in a solvent-free system was put forward which can ensure a high conversion aswell as high enzyme stability. We also prepared carrier-free immobilized lipase ascross-linked enzyme aggregates （CLEAs） and achieved the desired results whenapplied them to the synthesis reaction of flavor ester. The main conclusions in thisthesis were summarized as follows.Enzyme-catalyzed esterification for cinnamyl acetate synthesis: Cinnamylacetate was synthesized by lipase （Novozym435） through esterification route withacetic acid and cinnamyl alcohol in an organic solvent system. The optimum reac tionconditions were summarized as: substrate molar ratio （acetic acid/cinnamyl alcohol）=5/4, enzyme loading30mg （4.5U）, reaction temperature30oC. The conversion ofcinnamyl alcohol is more than88%under the optimum reaction conditions. Throughthe optimization of enzyme recovery step, the immobilized lipase （Novozym435） inthis system can be reused four times and all cycles kept more than74%conversion ofcinnamyl alcohol.Enzyme-catalyzed transesterification for cinnamyl acetate synthesis: Thesynthesis of cinnamyl acetate was successfully catalyzed by lipase （Novozym435）through transesterification reaction using ethyl acetate as the acyl donor in asolvent-free system. Under optimized reaction conditions (40oC,2.67g·L^-1of enzymeloading, ethyl acetate/cinnamyl alcohol=15:1,3h),90.06%of conversion wasobtained and the immobilized lipase can be reused at least6cycles with little declineof activity. Compared with the esterification system, the transesterification has moreadvantages both in the conversion of cinnamyl acetate and the reusability of theimmobilized lipase.Preparation and application of lipase CLEAs: The Candida antarctica lipaseCLEAs was prepared using100%saturated ammonium sulfate as the precipitatingagent and80mM glutaraldehyde as crosslinking agent after6h crosslinking reaction.The optimum temperature and pH for carrier-free immobilized lipase is much betterthan the free enzyme. When applied the lipase CLEAs in the synthesis of cinnamyl acetate with ethyl acetate and cinnamyl alcohol, the final conversion of cinnamyl was84%, and the residual activity of CLEAs was more than80%after6times used.The reaction kinetics of lipase-catalyzed transesterfication: A kinetic modelwas established based on the classical Ping-Pong Bi-Bi mechanism as well as theexperimental data. The kinetic parameters were obtained through fitting the kineticdata with the NL fit module in Origin8.0: Vmax=41.807mmol·L^-1min^-1，KA=2.241mmol·L^-1，KB=206.82mmol·L^-1，KiB=0.461mmol·L^-1.