介孔材料组装酶蛋白的研究
摘要
本论文对介孔材料组装酶蛋白进行了较为系统的研究,首先合成了不同孔径的介孔材料并进行了表面修饰,以介孔材料为载体进行了多种酶的组装的研究。对固定方法,酶蛋白固定量、催化活性、固定化酶的性质和稳定性进行了深入的研究。其中,首次用冷冻-真空吸附法对酶蛋白进行了成功组装,为介孔分子筛组装酶蛋白提供了一种新的实验方法,该方法在控制合适真空度的情况下,在保持酶活性的同时,组装量巨大,远远高于浸渍法及其它常见方法。首次运用能谱手段对酶蛋白在介孔材料上的固定位置进行表征,结果表明透射电镜能谱和扫描电镜能谱联合进行固定化酶活性定位这一手段完全可行,解决了由于杂蛋白、杂质进入孔道而造成的酶蛋白用普通手段,如N2吸附、UV 光谱等手段无法表征的难题。对在工业上应用较大的青霉素酰化酶、脂肪酶进行了固定,尤其在稳定性方面进行了深入研究,研究发现固定化酶的稳定性与介孔材料的孔径关系较大,当介孔材料的孔径与酶分子的大小接近时,稳定性较高。通过对介孔材料的修饰,用交联法固定时,固定化酶的操作稳定性好,可多次反复应用,显示了较好的工业应用前景。利用介孔材料固定化酶稳定性高的优点,本论文最后进行了非水催化的研究,首次在有机介质中成功进行了肽合成,并发现介孔材料的孔道表面性质对合成影响巨大。通过介孔材料固定辣根过氧化物酶成功进行了高分子聚合物聚苯胺(PANI)的合成,得到结构特殊的邻位聚苯胺纳米棒和管。
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 the
active 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’s
ability 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.
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 the
active 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’s
ability 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.
引文
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