Study On The Application Of Lipase And Its Immobilization Technology In The Preparation Of Typical Acyclic Monoterpene Acetate

Acyclic monoterpenes acetate has a great value of application, because it has been widely applied in the food, beverage, cosmetics and pharmaceutical industry as an important kind of flavor and fragrance component. Considering the high cost of natural extraction method, pollution and byproducts of traditional chemical synthesis, the aim of this dissertation is to prepare two kinds of typical acyclic monoterpenes acetate, or citronellyl acetate and geranyl acetate, by the reaction of lipase-catalyzed transesterification in non-aqueous phase. The main contents of this work were as follows:(1) Preparation of citronellyl acetate and its reaction kinetics:Six free lipases from different microbial origins were initially screened,1# lipase was identified as the optimal enzyme for the experiments. The effects of various factors were studied to establish a more efficient biosynthesis system. The result indicated that the maximum reaction conversion occurred when vinyl acetate was employed as both acyl donor and solvent. The optimal reaction temperature was 40℃. The external diffusion limitation could be greatly reduced by increasing the agitation speed to 200rpm. The enzyme load was 9mg/mL. Under the optimized reaction conditions, the yield can reach 99% after 5h. Substrate inhibition was absent when the substrate concentration was below 500mmol/L, but the experimental results indicated that the product inhibition effect should be considered. Operational stability research of lipase revealed that the yield reduced to 55.57% rapidly after two batch cycles, which indicates that the operational stability was not very well. The kinetics of the lipase-catalyzed transesterification synthesis of citronellyl acetate in a solvent-free system suggested that the current model should be based on the ping-pang bi-bi reaction mechanism in the presence of the inhibition of citronellyl acetate. The rate equation was expressed as: From the experimental data, Matlab was used to simulate the optimal model parameters, and the results were as follows: Vw=0.4563mmol/L/min KA=1.408×103mmol/L KB=0.5336 KQA=2.318×10-3 KBQA=2.038×10-2L/mmol KIQ=5.001×102mmol/L The model parameters were substituted into the rate equation, thus the relative error of the model was calculated to be 11.98%, and the experimental values could be satisfactorily fitted to the simulated values.(2) Preparation of geranyl acetate and its reaction kinetics:After screening six free lipases from different microbial origins,1# lipase was identified as the optimal enzyme for the experiments. The optimal experiment conditions were determined as follows:vinyl acetate was employed as both acyl donor and solvent, the optimal reaction temperature was 30℃, the agitation speed was 240rpm, and the enzyme load was 12mg/mL. Under the optimized reaction conditions, the yield can reach 99% after 3h. Substrate inhibition was absent when the substrate concentration was below 500mmol/L, and the product inhibition effect can also be ignored. The yield reduced to 42% after eight batch cycles, which indicates that the operational stability need to be improved. A kinetic model based on the ping-pong bi-bi mechanism without inhibition by geraniol and geranyl acetate was proposed. The rate equation was expressed as: The experimental data was simulated and the parameters were as follows: Vm=0.1154mmol/L/min KA=1.886×102mmol/L KB=0.7842 KQA=0.1335 KBQA=1.400×10-3L/mmol The relative error of the model was calculated to be 1.88%, and the experimental values could be satisfactorily fitted to the simulated values.(3) Immobilization of lipase:Poly-o-phenylenediamine of hollow particles which was synthesized by o-phenylenediamine as the monomer was applied as the carrier to immobilize 1# lipase by the adsorption method. The carrier was modified by the silane treatment. The surface modifications had great influences on the bound protein and activity of the immobilized-lipase. Among them, the adsorbed lipase onto N-octyltrimethoxysilane modified carrier showed highest activity. The immobilization conditions including the activation degree of the support, the mass ratio of lipase to the support, the pH of the buffer, the concentration of the buffer, adsorption time, adsorption temperature and agitation speed were investigated and the optimum values were 8mL,1/30, pH8.0,40mmol/L phosphate buffer,5h,30℃ and 200rpm, respectively. It was shown that the immobilized-lipase yielded a specific activity of 11.8 and 12.6 folds of the free lipase in hydrolysis and transesterification reaction, respectively. In addition, the thermal stability and storage stability of the immobilized-lipase were also improved obviously.(4) Application of immobilized-lipase in preparation of typical acyclic monoterpenes acetate:The immobilized-lipase was used to catalyze synthesis of citronellyl acetate and geranyl acetate. The optimal conditions of two reaction system were determined as follows:The amount of immobilized-lipase was 0.4g, the reaction temperature was 50℃, the agitation speed was 200rpm. Under the optimized reaction conditions, the yield of citronellyl acetate and geranyl acetate both can reach more than 99% after 1.5h. In the preparation of citronellyl acetate, the immobilized lipase exhibited higher stability than the free form and retained 98% of the original activity, while the free enzyme left 56% after two batch cycles. Moreover, in the preparation of geranyl acetate, the immobilized lipase exhibited higher stability than the free form and retained 90% of the original activity, while the free enzyme left 50% after eight batch cycles.