The Studies On Encapsulation Of Enzyme In Mesoporous Materials

Silica mesoporous materials with narrow pore size distribution, as carriers in enzyme immobilization, have attracted increasing attention. The internal surfaces of these pores, which can achieve some 1000 m 2 g -1, are lined with silanol hydroxyls. Such feature may promise high potential as supports for catalytic applications. The immobilized enzyme on solid supports has been widely used in many investigations. When the enzyme is immobilized, its autolysis is minimized and protein aggregation is reduced. The immobilized enzyme offers several advantages, such as repeated use of enzyme, ease of product separation and improvement of enzymatic stability and so on. Previously, Balkus and his co-workers had immobilized enzymes onto mesoporous silicate materials, while they had been only successful in immobilizing small enzymes, because the mesoporous materials were still restrictive due to the limit of their pore diameters (ca. 20~60 ?). Little had been written about big enzymes and no systematic research had been done on the application of mesoporous materials as new support. In out work, we synthesized, for the first time, mesoporous materials of various diameters(6-22nm)which were used as carriers in the immobilization of many kinds of enzymes. And detailed studies were conducted on immobilization methods, the immobilized amount and the characterization of immobilized enzyme, etc. The characteristics and stability were also included in our research. It was found that the amount of enzyme immobilized in mesoporous materials was far larger than that in other carriers. The reason lies in the great surface area of these materials. Vacuum adsorption was also first employed in the protein immobilization. The results indicate that this method can guarantee a large amount, achieving 400mg/g support,which is incomparable for other means. As long as vacuum degree is properly controlled, the enzymatic activity can retain. The characterization of enzyme on site of mesoporous material was carried out via EDS. The results show that the localization of the activity of immobilized enzyme through TEM EDS and SEM EDS is perfectly applicable in that it can provide the solution than other methods to the problem of characterization caused by the entry of other protein and materials into the channels. Penicillin G acylase and lipase, industrially promising, were also attempted through immobilization. Specifically a study was carried out in its stability. Penicillin G acylase is globular enzyme of a large size (100 ?) and molecular mass (123,000). So it is difficult to be driven into the pores of mesoporous solids for MCM-41 (30 ?) and SBA-15 (60 ?). The usage of swelling agent, such as TMB, made it possible to synthesize materials with large pore size. At the same time, we also concentrated on the largest pore mesoporous solid for further study, because it could permit easier access of reactant molecules to theactive sites of the enzyme and the transport of products out of the pores. Penicillin G acylase catalyses the cleavage of the amide bond in the benzyl penicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. The activity of nature PGA is low, which limits application of PGA. By immobilizing PGA on a solid support, it can be reused and its useful lifetime be extended because the immobilized PGA is less susceptible to degradation, aggregation, or denaturation. In our work, we had examined the efficacy of pure silica mesoporous material in immobilizing the enzyme by physical adsorption. However, there was very significant leaching observed from the support under the reaction conditions and it was, therefore, concluded that the interaction between the enzyme and the inorganic surface was not strong enough. One approach to reducing the degree of leaching of enzymes from mesoporous material is to functionalize surface of mesoporous material. So from the perspective of pharmaceutical industry, it is practical to immobilize enzyme by crosslinking or covalent attachment in order to prevent enzyme from leaking and facilitate the reuse of the immobilized enzyme. Once again, triethoxysilane with a NH2 group is usually the reagent for this type of experiment. Amines can couple enzyme molecules with support by using glutaraldehyde as coupling agent through Schiff reaction. The research results indicate that the functionalization of the internal surfaces of mesoporous solids permitted enzyme immobilization. Catalytic testing of the most promising composite supported enzyme solids showed that strong attachment did not adversely affect the enzyme'sability to hydrolyse penicillin. This solid achieved a catalytic activity per gram of original protein 84% of that measured for the free enzyme. Re-use of the catalysts is also possible. Retention of up to 90% of the PGA is observed in the most favorable cases after used 20 times. The surface characteristics of the mesoporous silica, and matching of the sizes of the enzyme molecules and the pore diameters of the mesoporous silica are essential for the stabilization of enzymes. Lipase is an interesting enzyme with potential application in a number of industrial processes such as the synthesis of triglycerides and esterification of terpenic alcohols. The immobilization of lipase resembles that of PGA in outcomes. These characterizations show that immobilized enzyme in mesoporous materials can be applied in practical production. α-chymotrpsin immobilized in SBA-15 was employed as a biocatalyst in the synthesis of protected peptides, BOC-Phe-Arg-Ome, in ethyl acetate media. The influences of reaction conditions such as pH, temperature, reaction time and channel property of SBA-15 on the enzymatic peptide synthesis were studied and optimized. The results show that the hydrophobilic environment of the inner surface of SBA-15 channel effects significantly on the enzymatic peptide synthesis. These results also indicate that the immobilized α-chymotrpsin is an effective catalyst for peptide synthesis in organic media. At last,the oxidative polymerization of aniline was carried out in the channel of mesoporous material SBA-15 in the presence of horseradish peroxidase (HRP )in a mixture of NaAc buffer and organic solvent and ortho-directed polyaniline (PANI) with special structures in polymerization occurring in controlled nanoscale are obtained.