The Research On Catalysis Performance And Stability Of Enzyme Covalently Immobilized Onto The Surface-modified TiO₂Microspheres

Titania （TiO₂） has many advantageous properties such as non-toxicity, highmechanical strength, high thermal and chemical stability, anti-microbial degradationand resistance to organic solvents, etc. It has been found that catechol and catecholicderivatives could chelate with titania to form a stable salt chelate complex at roomtemperature rapidly. Inspired by the chelation between catechol and titania, in thisstudy, enzymes were attached onto the titania surface via the chelation and covalentbinding.The details in this study were summarized as follows:Firstly, titania microspheres （500⁶00nm） were synthesized by a modifiedsol-gel method. The titania microspheres were surface modified with carboxylic acidgroups through a facile chelation method by using3-（3,4-dihydroxyphenyl） propionicacid as the chelating agent. The catalase and lipase were covalently immobilized ontothe titania surface successful via EDC/NHS coupling reaction. The results of Fouriertransform infrared spectra （FTIR） indicated that titania was surface modified by3-（3,4-dihydroxyphenyl） propionic acid and the enzymes were covalently attachedonto the functionalized titania successfully. The catalysis performance and stability ofimmobilized enzymes were investigated and compared with those of free enzymecounterparts. The relative activity of immobilized catalase and lipase were65%and60%respectively. The loading ratio of immobilized catalase and lipase were150mg/gsupport and200mg/g support respectively. The kinetic parameters of two enzymeswere changed a little after covalent immobilization.Secondly, titania microspheres were surface modified through the facilechelation method by using dopamine as the chelating agent. Then, catalase and lipasewere covalently attached onto the titania microspheres surface using glutaraldehyde ascross linking agent. FTIR indicated that the covalent bonds between the titania andenzyme molecular were formed. Then, the catalysis performance and stability of theimmobilized enzymes were investigated too.Thirdly, the catalase was modified with3-（3,4-dihydroxyphenyl） propionic acidvia EDC/NHS coupling reaction. Then, the catalase was attached onto the titaniamicrospheres through the facile and rapid chelation between the titania andcatechol-based group. The relative of immobilized catalase was60%, while the loading ratio was up to500mg/g support. In order to check the universality of thisimmobilization method, the alcohol dehydrogenase was immobilized onto the titaniamicrospheres via the same method. The relative activity of immobilized alcoholdehydrogenase was up to90%. The FTIR indicated the enzymes were covalentlyimmobilized onto the titania microspheres.Finally, we compared the catalysis performance and stability of immobilizedenzymes prepared by different methods, such as, loading ratio, relative activity,kinetic parameters, thermostability, recycling and storage stability. The merit andshortcoming were summarized through this comparison.