Study On The Immobilization Of Aminoacylase By Polyamidoamine (PAMAM) Dendrimer

Dendrimer is a new type of functional macromolecule compound whose specialties are to design the molecular size, shape and functional group at the molecular level. The introduction of PAMAM can improve the surface efficiency. Enzyme immobilization offers advantages over free enzymes in stability, continuous processes, reusability of catalysts, ease of enzyme survival in the reaction mixture and adaptability to various environment designs. Effective immobilized technology and ideal supports were necessary for the enzyme immobilization.In this paper, Divergent synthesis was used as the synthesis process of the PAMAM surface modified silica. The silica used is of 50μm in diameter. After Silanization by 3-aminopropyltriethoxysilane (APTES), 3.0G PAMAM was grown from the amino groups by repetition of two steps under certain conditions: Michael addition of methyl acrylate (MA) to amino groups on the surface and amidation of the resulting terminal ester groups with ethylenediamine (EDA). The molecular structures and properties of the product have been characterized by terminal group analysis, nuclear magnetic resonance (NMR), scanning electron microscope (SEM), fluorescence microscope (FM) and elemental analysis (EA). The result demonstrated that the structures and properties of the synthesized dendrimers was in line with the theoretical structures and properties.Glutaraldehyde was used to crosslink PAMAM modified silica with aminoacylase, using 1% glutaraldehyde to crosslink PAMAM modified silica and aminoacylase in 25℃for 4h. The performances of the immobilized enzyme were evaluated by the separation of N-acetyl-DL-methionine. The properties of immobilized enzyme were also studied. Various characteristics of immobilized aminoacylase, such as pH, concentration of Co²⁺, concentration of substrate, are considered. The optimized conditions of immobilized aminoacylase were found to be pH 7.5 and 30℃respectively. The thermal stability and storage stability were also valuated. The results indicated that the immobilized enzyme had higher thermal and storage stability than the free enzyme. Half-life of the immobilized enzyme was 34d.Immobilized aminoacylase was used to evaluate the performances of the immobilized enzyme by capillary zone electrophoresis (CZE). The results showed that the immobilized enzyme had good efficiency, reproducibility and stability.