Study On The Preparation Of Homogeneous Macroporous Materials And Their Application In The Immobilization Of Glucoamylase

At present,immobilization of enzymes is an attractive research field.Immobilized enzyme can overcome the shortages of free enzymes,such as poor stability,poor reusability and difficulty in separation.The carrier and enzyme immobilization method have an important influence on the activity of the immobilized enzyme.The homogenous macroporous structure can provide optimum mass transfer for macromolecules.This paper aimed to develop new methods and supports for the immoblization of enzymes with high mass transfer performance,high activity and stability.Immobilization of glucoamylase,which is widely used in industry,is used as a research model to prepare homogenous macroporous materials with high porosity,high surface area,and high mechanical strength.The specific contents are as follows:?1?Using PS colloidal crystal as the template,silica sol as the precursor,ordered macroporous thiol group functionalized silica hybrids?3DOM SiO₂-SH?was first prepared.Then,ordered macroporous carboxyl group functionalized polymer@silica composites?3DOM SiO₂-COOH?were prepared by grafting a polymer layer onto the pore walls in the3DOM SiO₂-SH.Acrylic acid was used as the functional monomer and glycerol1,3-diglycerolate diacrylate as the cross-linker.The pore structure was characterized by FTIR and Raman spectrometer,SEM,Brunauer-Emmet-Teller measurement,DSC and TGA.The results show that the 3DOM SiO₂-COOH had a uniform interconnected macroporous structure,a high porosity and a high mechanical strength.Batch adsorption tests towards lysozyme show that the adsorption performance was good and the adsorption process was apparently dominated by the internal diffusion.Several isothermal adsorption and kinetic models were applied to discuss their adsorption be haviors.The results show that the inferred parameters such as maximum adsorption amount?q_m?,adsorption equilibrium constant?K_l?,diffusion coefficient?D_e?and adsorption energy?E?were similar for the particles range from 65 to 225?m.However,for the large particles of 550?m,such parameters were different from the smaller particles due to the deep sites being not completely accessible for the proteins.?2?AfteractivatingwithN-?3-Dimethylaminopropyl?-N?-ethylcarbodiimide hydrochloride and N-hydrorysuccinimide,3DOM SiO₂-COOH materials were used as supports to immobilize glucoamylase by a covalent bonding method.The results show that the immobilized enzyme homogeneously distributed in the 3DOM materials.The optimum pH of immobilized and free enzymes was both at 5,and the optimal reaction temperature was at 55?.Michaelis constants of the immobilized enzyme and free enzyme were 3.78g·L^-1 and 3.97 g·L^-1,respectively.The immobilized enzyme presented higher thermal,pH,storage stabilities and higher reusability compared with the free enzyme.The results indicate that 3DOM SiO₂-COOH could be a novel support to immobilization of enzymes.?3?Glucoamylase encapsulated polyacrylamide cryogels were prepared by combination of the in-situ physical embedding method and the freezing polymerization method.Acrylamide was used as the monomer,N,N'-methylenebisacrylamide as the crosslinking agent,N,N,N',N'-tetramethylethylenediamine as the accelerator,potassium persulfate as the initiator,and ice crystal as the porogen.These gels have high porosity,large pore size and high mechanical strength.And the immobilized enzyme has good stability.After reusing for 22 days,the activity retained 71%of the original immobilized enzyme.The results show that the novel free zing polymerization-embedding method has a promising application in immobilization of enzymes.In summary,3DOM SiO₂-COOH can be used for enzyme immobilization,protein adsorption and separation.Glucoamylase encapsulated polyacrylamide gels prepared by freezing polymerization-embedding method have high activity and reusability.The results have good scientific and practical values.