Research On Immobilized Lipases Catalyzing α-pinene Epoxidation

α-pinene is one of the common monoterpene,widely distributed in aromatic plants like pine tree,mint and so on.Its corresponding epoxide is an important intermediate and has been widely used in the fields of flavor,medicine and pesticide.Traditional chemical epoxidation of α-pinene has many disadvantages,such as low yield,lots of by-products and difficult separation for catalysts.Therefore,improvement and optimization of the epoxidation is urgently needed to meet with the new concept of "green and sustainable development".Chemoenzymatic epoxidation has the advantages of low reaction temperature,few byproducts and high selectivity,and has a promising application and good research value.In this paper,the problems of the the biocatalyst easily deactivating in the epoxidation were studied from the aspects of the optimization of the reaction system and the preparation of the immobilized catalyst.In this thesis,the optimization of the reaction system was firstly explored.Using the commercial immobilized lipase Novozym435 as a catalyst and isopropanol,a water-miscable solvent,as reaction medium,the system included α-pinene,ethyl acetate and hydrogen peroxide to form one-phase reaction system.The effects of concentration of hydrogen peroxide,ethyl acetate and acid trapping acids on epoxidation of α-pinene were investigated.The results showed that the enzymatic performance in the single-phase system was better than that in the traditional organic/water "two-phase" reaction system.Under the optimal conditions,the conversion of epoxidation reached 69.5% and the yield reached 61.2% in 90 min.Besides,the reaction rate was significantly higher than that reported in the literature.Moreover,the relative acitivity of Novozym435 remained 57.5% after 6 runs.In addition,the system was also used for the chemoenzymatic epoxidation of alkenes such as styrene,cyclohexene and 1-octene and showed universality in some extent.It was considered that,in the traditional organic/water "two-phase" reaction system,the enzyme easily deactivatied by high concentration of hydrogen peroxide and the mass transfer limitation at the phase interface were overcomed,thus improving the enzyme activity and stability.In order to improve the performance of the catalyst,the immobilization of lipase CRL(Lipase from Candida rugosa)on MOF was attempted.The effects of initial p H value of 2-methylimidazole solution,material addition ratio,enzyme addition amount,aged time and drying procedure on the immobilized lipase by ZIF-8(CRL@ZIF-8)were investigated.The results showed that under the condition that the initial p H value of 2-methylimidazole solution was 8.0,the molar ratio of raw material was 1:4(zinc: 2-methylimidazole)and the amount of enzyme was 2.75 mg,the immobilized enzyme had the best performance with 1.08 U/mg of the specific activity and 18.4% of activity recovery.CRL@ZIF-8 showed the size selectivity to substrates.Using p-nitrophenyl acetate as substrates,it maintained 72.8% of relative activity after 9 runs.When used for epoxidizing α-pinene,the immobilized enzyme had only 20.7% of conversion in 180 min,which did not reach the expected results.The Lipase CALB(Lipase B from Candida antarctica)was immobilized by the above methods.This immobilized enzyme(CALB@ZIF-8)has excellent performance for α-pinene epoxidation,where the conversion and selectivity achieved 80.6% and 90.4%,repectively,in 90 min.To solve the ZIF-8 decomposition caused by acetic acid,the strategy of introduction of trisodium citrate into the immobilized enzyme was carried out and then an immobilized enzyme(CALB@ZIF-8@Zn-BTC)with trisodium citrate was successfully obtained.The α-pinene epoxidation catalyzed by the immobilized enzyme achieved 82.2% conversion and 95.0% selectivity in 90 min.The stability of the catalyst was significantly enhanced,which achieved 67.9% of relative activity and 70.6% selectivity after 7 runs at a concentration of 1 mol/l hydrogen peroxide.EDS analysis showed no significant loss of trisodium citrate in immobilized enzyme.This immobilized lipase provided a positive reference for preparation of biocatalysts for chemo-enzymatic epoxidation.