Immobilization Of Lipase On Monolithic Macroporous Silica-based Materails And Their Application

Lipase is a kind of acylhydrolase with interfacial activity.It has high catalytic activity on the interface of water and oil and can catalyze the hydrolysis of lipids,the synthesis of esters,transesterification,ammonolysis and acidolysis.It has been widely used in many industrial fields.The enzymatic kinetic resolution in the preparation of chiral compounds is based on the high activity,high stereoselectivity and substrate specificity of lipases in organic solvents,and the transesterification with a single enantiomer to achieve the resolution of racemes.The obstacle in the industrial use of free enzyme is that it is difficult to recycle and reuse and can easily cause secondary pollution of the product.Therefore,immobilization of lipase is an effective way to facilitate the recovery and reuse of lipase.Silica-based porous materials,as a kind of inorganic porous materials,have good thermal stability and mechanical stability.In this paper,a monolithic macroporous SiO2 material with high porosity,high specific surface area and uniform pore size was prepared by hard template method.It was modified by different methods for lipase immobilization as carrier,and the immobilized lipase was used to chiral resolution of 1-phenylethanol.The results are as follows:?1?The monolithic silica was prepared by using an epoxy resin macroporous polymer with pillared structure as template.Pore structures and surface properties of the as-prepared materials were characterized by SEM,MIP,FTIR and N₂ adsorption-desorption.The results show that the macroporous silica has 3D continuous pass-through macroporous structure;its pore wall is constructed by continuous silica nano-film and has abundant mesopores.The specific surface area and porosity are 75.1 m²/g and 92.3%,respectively.Candida rugosa lipase?CRL?was immobilized on the macroporous silica by adsorption method.The effects of the initial CRL concentration,pH and immobilizing time on lipase immobilization were investigated,and the properties of free and immobilized lipase were studied.The activity of immobilized lipase reaches 4825 U/g under conditions of initial CRL concentration 0.6 mg/mL,pH of 8.0 and immobilizing time of 10 h.Compared with free CRL,the pH,thermal and storage stabilities of immobilized lipase are improved significantly.The immobilized lipase retains 68%residual activity after 8 consecutive operations.?2?Epoxy resin was adhered on the surface of the macroporous SiO2 pore wall by solvent evaporation method to prepare the composite macroporous material EP/SiO2 and then EP/SiO2 was used to immobilize Pseudomonas fluorescens lipase?PFL?.The immobilization conditions of lipase were optimized.The activity of the immobilized lipase?PFL@EP/SiO₂?reaches 5530 U/g under conditions of initial PFL concentration 0.8 mg/mL,pH of 8.5 and immobilizing time of 12 h.Compared with free lipase,the pH,thermal and storage stabilities of immobilized lipase are improved significantly.The immobilized lipase retains 64.9%of residual activity after 10consecutive operations.?3?The chiral resolution of 1-phenylethanol was carried out by PFL@EP/SiO2.The effects of reaction time,solvent,water activity,and catalyst dosage and substrate/acyl donor molar ratio on the resolution of 1-phenylethanol were investigated.The results show that the water activity and the amount of catalyst have great influence on the reaction.When the reaction time is 48 h,the solvent is cyclohexane,the CuSO4?5/3 hydrated salt pair is used to control the water activity of the reaction system,the amount of catalyst is 0.15 g and the molar ratio of substrate to acyl donor is 1:3,the ee_s value of the transesterification of?R,S?-1-phenylethanol with vinyl acetate catalyzed by PFL@EP/SiO₂ was 99.5,and ee_p>99.5.The conversion rate was 49.9%and the E value was greater than 2000.?4?Poly?GMA-co-Trim?/SiO2 composite macroporous material was prepared by in situ polymerization of glycidyl methacrylate?GMA?and trimethylolpropane trimethacrylate?Trim?in the channels of the macroporous SiO₂.PFL was immobilized on Poly?GMA-co-Trim?/SiO₂ and the immobilized lipase was used for chiral resolution of 1-phenylethanol.The effects of reaction time,solvent,water activity,and catalyst dosage and substrate/acyl donor ratio on the resolution reaction were investigated.When the reaction time is 36 h,the amount of catalyst is 0.20 g,the molar ratio of substrate to acyl donor is 1:4,cyclohexane is used as solvent and the water activity of the reaction system is regulated by adding CuSO4?5/3.The eep and ees values of the transesterification of?R,S?-1-phenylethanol with vinyl acetate catalyzed by the immobilized enzyme was 97.7%and 90.1%,respectively.The conversion rate was 48%and E value was more than 200.