【作者】 吴卓夫；

【导师】 李正强；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 在酶工程的众多技术中,酶的固定化技术占有重要的位置。但是现有的固定化技术都有各自的优势及不足,寻找具有普适性的固定化技术路线是该领域的工作者们所关心的重点。2006年本组构建了“fish-in-net”固定化技术,该方法所形成的硅基笼孔结构适用于固载较多种类的酶。在本文中,我们首先就“fish-in-net”固定化技术中所存在的基础理论问题展开研究:(1)利用具有笼孔结构的SBA-16硅基分子筛为载体,检测肌红蛋白的吸附速率,探讨在笼孔结构中蛋白质分子的运动行为;(2)利用SBA-15硅基分子筛为载体吸附胰蛋白酶,利用数学公式讨论pH变化对硅基介质与酶之间相互作用的影响,探讨固载率与固定化酶活力之间的规律;(3)利用氨基化的SBA-15为载体,完成对孔道内肌红蛋白及孔道外周溶菌酶的双酶固载,摸索修饰后硅基介质在双酶固载中应用的可行性。在此基础之上,我们将“fish-in-net”技术与分子印迹技术组合起来,通过“稳定化-固定化”的思路,制备具有高活力的固载乳糖酶。而后,在“fish-in-net”技术所形成的硅基介质表面连接溶菌酶,赋予笼孔结构的载体以双酶特性。我们的研究表明:(1)相比于常规直筒型的结构,在“fish-in-net”技术所形成的笼孔结构中蛋白质分子的运动速率相对较慢,蛋白分子与硅基骨架之间的相互作用较弱,蛋白分子处在一种更为自由的状态;(2)固载于硅基介质中的酶的活力随固载率的升高而升高,达到一定值后随固载率的升高而下降;(3)当完成硅基介质孔道内第一种酶的固载后,通过修饰于硅基介质外周的活性氨基可以在其外周连接第二种酶,且两种酶之间并不相互影响彼此的活性。后续的实验证实:通过将“fish-in-net”技术与分子印迹及共价连接法组合,能够在笼孔结构中得到具有高活力的固载乳糖酶,在孔道外共价连接具有抑菌功效的溶菌酶。更多还原

【Abstract】 The merits of the enzyme used as biocatalyst are as follow: high catalytic efficiency, strong specificity, catalytic activity modulated by its ligand. However, the enzyme is susceptive to its surrounding environment change. Once the enzyme is placed in the environment which contains strong acid, strong base, high temperature, high ionic strength and some organic solution, the catalytic activity of the enzyme may decrease or completely lose. Moreover, the enzyme in homogeneous phase reaction system is not more easily separated as compared to the traditional chemical catalyst in heterogeneous phase reaction system. In order to overcome above depicted problems, the immobilization of the enzyme usually is adopted to fulfill the broad application in food industry, medicine, fine chemistry and diagnosis area.The immobilization technologies have been attracted attention in early 20th century. Its usually are consisted by physical adsorption, chemical covalent, microcapsule entrapment and sol-gel encapsulation. However, the people find out that how to rationally integrate all kinds of technologies to achieve enzyme immobilization is still a difficult problem.As compare to other materials, advantages of silica-based matrix are as follows: good hydrothermal stability, high mechanical stability, resistance against biodegradation, biocompatibility and low toxicity etc. Hence, we have successfully encapsulated several kinds of enzyme (fumarase, trypsin, lipase, and porcine liver esterase) into silica-based matrix with cage-structure using“fish-in-net”route in 2006. Obtained encapsulated enzyme exhibited perfect operation stability and higher conversion (%) of the substrate.“fish-in-net”route was applied to the immobilization of many kind of enzyme.In this paper, we devoted to study the basic theory of“fish-in-net”route and combine“fish-in-net”route with other techniques to endow it with perfect use value.In chapter 2, through hydrothermal method, a series of SBA-15 and SBA-16 were synthesized at 100℃, 160℃, 180℃and 200℃. The pore size was controlled within a certain range (5–17 nm). X-ray diffraction spectrum, N2 sorption isotherms and TEM images demonstrated that SBA-15 and SBA-16 possess 2D and 3D channel, respectively. Above mentioned mesoporous molecular sieve were used to adsorb myoglobin (17KD). Absorbency of myoglobin was detected by UV-VIS measurement in order to estimate adsorption of the protein and compare corresponding process depending on time. Adsorption experiment dates demonstrated that adsorption velocity of SBA-16 with hexagonal (p6mm) structure is lower than that of SBA-15 with cubic (Im3hm) structure. It is suggested that the interaction between the silica-based matrix and myoglobin become weaker in cage produced by“fish-in-net”route, and myoglobin possess more degree of freedom compared with in SBA-15.In chapter 3, SBA-15 was synthesized using non-ionic surfactant P123 as the template and tetraethyl orthosilicate as silicon source in acid media. Such material was used as a carrier to load trypsin. Through mathematical model simulation, optimal pH of immobilized trypsin was further confirmed. The catalytic ability, leaching and denaturation-renaturation capability of immobilized trypsin were studied in detail by using mathematical model simulation. The catalytic ability and reusability of trypsin were determined under any pH condition according to the model. The relation between loading amount and the enzymatic activity is surveyed. The leaching, denaturation-renaturation capability and optimal pH of immobilized trypsin were detected. Results showed that the special activity of immobilized trypsin decreases with the increase of loading amount when initial concentrations of the enzyme before adsorption were all equal and that the special activity of immobilized trypsin was similar to that of free trypsin when loading amount approach to minimum. With the increase of reaction time, the activity of free trypsin decreaseed and the activity of immobilized trypsin increased, indicating that stability of immobilized enzyme was improved and that degradation of free enzyme were inhibited. Optimal pH value of immobilized trypsin was 7.5, but the trypsin slightly leached out the matrix. After three cycles, 80% of initial enzymatic activity was obtained. In chapter 4, aminopropyl-Functionalized SBA-15 had been used as a carrier to absorb myoglobin. Then, lysozyme had been bound to amino groups of mesoporous materials with succinic anhydride and glutaraldehyde as coupling agent respectively to avoid leakage of absorbed myoglobin through the effect of steric hindrance. The properties of the matrix before and after adsorption were characterized by transmission electron microscopy, N2 adsorption/desorption, thermogravimetric analysis and UV-VIS measurements. With o-dianisidine and H2O2 as the substrate, the peroxidase activity myoglobin was assayed. With Micrococus Lysodeilicus as the substrate, antibacterial activity of lysozyme was assayed. Results demonstrated that the materials obtained not only present peroxidase activity of myoglobin but also offer antibacterial activity of lysozyme.In chapter 5,by using tetraethylorthosilicate as a silica resource and triblock copolymer P123 as a template, the encapsulation of ?-galactosidase with three different models of without protection, protection of protective agent and molecular imprinting technique pretreatment was accomplished through modified“fish-in-net”route at pH 5.0. The highest enzymatic activity of ?-galactosidase was gained by using pretreatment of molecular imprinting technique. Scanning electron microscopy (SEM) images showed that the matrix of encapsulated ?-galactosidase was made of an aggregation of uniform microspheres of 200-300 nm, and N2 adsorption/desorption isotherms demonstrated that the matrix of encapsulated ?-galactosidase possessed average Brunauer-Emmett-Teller (BET) pore size of 27 ? and narrow pore-size distribution. More importantly, compared with encapsulated ?-galactosidase without protection, the hydrolytic activity of encapsulated ?-galactosidase pretreated by molecular imprinting technology was about 3 times and 1.8 times, while the enzymatic activity of encapsulated ?-galactosidase with the protection of protective agent increased only 1.3-fold when lactose and o-nitrophenyl-β-D-galactopyranoside (ONPG) were used as substrates, respectively. The protective effect of molecular imprinting technique pretreatment on the enzymatic activity after encapsulation was better than that of protection of protective agent. In chapter 6, the active amino group was bound to outer surface of the silica-based matrix which was used as carrier to encapsulate ?-galactosidase pretreated by molecular imprinting technique with APTES as a aminopropyl-functionalized agent, and then the lysozyme was linked to the active amino group with glutaraldehyde as an acrosslinking agent. The obtained product exhibited perfect operation stability for the hydrolytic activity of ?-galactosidase and significant antibacterial effect. This work should become a theoretical basis for preparing low-lactose milk.更多还原