Lipase Immobilization And Its Applications In The Synthesis Of Chemicals

pase-catalyzed processes are receiving more attentions as a promising alternative in the synthesis of chemicals because of its improving reaction conditions, high selectivity of biocatalysts and eco-friendly. To develop an new and less expensive immobilization process for industial applications, the immobilization of lipase from Candida sp.99-125using natural fibrious materials as a support was investigated in this thesis, as well as its applications in the synthesis of some chemcals. As an active and progressive area of green chemistry and technology, ionic liquids(ILs) are becoming increasingly popular as "green" solvents for a wide range of applications in bio-synthesis. In this thesis, an enzymatic modification of fats and oils in ILs was also investigated. Some researches have been done as follows:1. Silk fabric as a film support for lipase immobilization:the effect of surface hydrophilicity/hydrophobicity on the enzymatic activity and stabilitySilk fibers in the form of woven fabric were used as a novel and inexpensive carrier for the immobilizaiton of lipase Candida sp.99-125by adsorption. Hydrophobic silk fibers functionalized with methyl groups were prepared by treating with amino-functional slicones. In this study we compare the hydrolytic and synthetic activity of lipases immobilized on two kinds of silk fabrics with different hydrophilic/hydrophobic properties. It was found that the lipase immobilized on the hydrophobic support exhibited much higher activity of both hydrolysis (1.6-2times higher) and esterification (3-4times higher) due to the interfacial activation as compared to its immobilization on the hydrophilic support (untreated silk). The lipase immobilized on hydrophobic supports also showed an improving stability in different organic solvents. The esterification behaviour in organic media with viarable water content and operational stability of immobilized derivatives were also compared. The lipase immobilized on the hydrophobic fiber could maintained a constantly high avitity at a15%(v/v) water content system.While the acitivy of immobilized lipase on native fiber showed a clear dependence on water content in organic media and dereased rapidly at high water contents(>2%). Furthermore, lipase immobilization on the hydrophobic support exhibited a significantly improved operational stability in esterification reaciton. After27batch reuses, a high esterification yield(97%) was still retained. The results in this work indicates that a hydrophobic surface of fabric fiber promotes the interfacial activation of lipase and the woven silk could be a potential material as an immobilizaion matrix for industrial process.2. Lipase immobilization by derectly adsorbing fermentation broths and its appliction in the synthesis of biodiesel by esterification of oleic acid and ethanol A simple and new lipase immobilization process has been developed by derectly adsorbing fermentation broths using silk fabrics as support. The effect of lipase activity units and pH value of fermentation broth and the immobilization conditions were studied. The fermentation broths with a lipase acitity range from1000-5300U/mL were used in this study. The lipase-catalyzed esterification of oleic acid with ethanol in a solvent-free system was examined to measure the activity of immobilized lipase. The results indicated the catalytic activity of immobilization lipase increased with the increase of lipase activity units in fermentation broth and repetitious operations of adsorption could increase the resue times of immobilization lipases. The fermentation broth with a pH range between4-7was suitable for the immobilization. Furthermore, using the immobilized lipase perpared by adsorbing fermentation broth as catalysts, the reaction for the synthesis of ethyl oleate in a solvent-free system was optimized. Ethanol was found to have a significant inhibition on the lipase activity, but that could be improved by the stepwise adding of ethanol. Adding molecuar sieves into the reaction system as water adsorbents also increased the esterification. Finally, under the optimized conditions, a90%conversion was obtain at30℃after24h reaction. The half-life of the immobilization lipase was over30days. 3. Production of2-ethylhexyl palmitate in a fixed-bed bioreactor using a lipase immobilized on silk fabricsAn enzymatic process using a fixed-bed bioreactor with recirculation has been investigated for the scale-up production of2-ethylhexyl palmitate by esterification. the lipase from Candida sp.99-125was immobilized on a fabric membrane by adsorption. The fixed-bed bioreactor system consisted of a glass column (L,280mm and ID,50mm) equipped with a water jacket and packed with a spiral-wound sandwich of fabric membrane and stainless steel mesh. The water produced by the reaction was removed in a second column filled with molecular sieves and connected in series with the reactor. A maximum esterification degree of98%was obtained when the molar ratio of2-ethyl hexanol to palmitic acid was1.3. Adding molecular sieve decreased the water content at the reactor outlet and led to an increase in the rate of esterification. The effect of flow velocity on the esterification rate was found to be negligible. Long-term stability of the reactor was tested by continuing the reaction for30batches, keeping an average esteriflcation degree of about95%. This new type of immobilized lipase bioreactor is suitable for scale-up production of2-ethylhexyl palmitate.4. Synthesis of dicarboxylic esters by immobilization lipaseThe esterifications between3different disbasic acids (sebacic acid, azelaic acid, adipic acid) and11n-alcohols with carbon chain length ranged from (C4-C18) have been carried out in a hexane system using immobilized lipases from Candida sp.99-125as a catalyst. The results indicated that sebacic acid and azelaic acid had better reactivities than adipic acid with all the alcohols tested. As a model reaction, the esterification between sebacic acid and2-ethyl hexanol was optimized The effect of reaction temperature, molar ratio of substrates, enzyme loading, organic solvents and water controlling were studied. Under a optimized condition:lmmol sebacic acid,3mmo12-ethyl hexanol,5mL hexane, enzyme loading1000U,0.5g silica gel as the water absorbent,40℃,98%esterification was obtained after24h reaction. The immobilization lipases were reused40batches without obvious loss in its activity. More than10kinds of dicarboxylic esters were synthesized under the same optimized conditions and over95%esterfication were obtained in all cases.5. Structures of Ionic Liquids dictate the conversionn and selectivity of enzymatic glycerolysis:Theoretical characterization by COSMO-RSLipase-catalyzed glycerolysis of triolein has been examined using a group of tetraammonium-based ionic liquids (ILs) as media, specifically with functional groups in cation part. The results demonstrated that the reaction evolution and profile specificity of respective IL system could be quantitatively associated with the structural characteristics of the IL by means of quantum chemical and COSMO-RS calculation. Misfit interaction, Van der Waals interaction and chemical potential, etc. derived from COSMO-RS calculation are shown to be effective measures to delineate multiple interactions of ILs and then can be used to understand the effects of ILs on reactions. The hydrophobic substituents in the cation are found to contribute to the increase of triolein solubility and enhancement of initial reaction rate; while strong polar an ion and polyethoxyl and free hydroxyl groups in the cation part dictate improved product selectivity through reducing activity coefficients of monoglycerides. Integration of these structures into the same molecule constitutes a promising group of ILs that could produce over90%monoglyceride with almost100%triglyceride conversion, as well as bulky productivity, of particular potential for industrial applications. Overall, this work has presented a first attempt to characterize the IL structure-dependency of reaction specificity by associating structural variations of ILs with thermodynamic property changes of resided compounds and subsequent effects on reaction specificity. This might be of general value to help to understand the multiple solvation interaction among IL reaction systems at molecular level and promote the application of IL-mediated reactions to practical interests.