| After immobilized enzyme was successfully employed in industrial application by Japanese corporation Tanabe Seiyaku in 1969, enzyme immobilization is increasingly used in practice operation for its good characteristics such as stability and reusability. Enzyme immobilization can not only enhance enzyme stability and reusability but also improve its specificity and the enzymatic activity which lead to changes of its characteristics so that it really met the requirements of human beings. So far, some new enzymes have been invented.In the field of immobilized enzyme, various carrier materials are developed. Ingenious designed reactors possess the property of automatic operation in production process and decrease product cost at the same time. They hold different characteristics. There are application examples in some fields including food industry, fine chemical industry, pharmaceutical sector, environmental protection and development of new energy.However, the number of immobilized enzyme method put into production is few. The reasons are as follow: firstly, the cost of immobilization is so high that application in a large scale is limited; secondly, microenvironment affects enzyme's structure in the process of immobilizing. Micro-environmental change of the enzyme results in the loss of its activity and the decrease of immobilizing efficiency. According to problems above depicted, our goal in this experiment is devoted to investigate effects caused by micro-environmental change on enzyme."fish-in-net"encapsulation had established by our team in 2006, it means that the enzyme is added to encapsulation system during encapsulation in order to make cages as the reaction chamber, liking fishing in the net, and that confine these molecules in special spaces. A new way of developing an efficient biological catalytic reactor is invented. The enzyme, as the template as well as the core for catalysis, combines advantages of native enzyme with multi-channel feature so that chemical environment of the enzymes in carrier is similar to that of native enzyme in the aqueous solution. This is beneficial for protein rotation and conformational transitions and provides for high catalytic activity. Immobilized enzyme prepared in this way has good catalytic activity, persistent stability and high repetition.However,"fish-in-net"route has some underlying problems. Proteins may escape from uniform mesoporous channels when its size is smaller than the diameter of the channel. In order to study completely above mentioned problem, we need to simplify study system. Based on the study of the escape situation, we created a new concept of"fish-in-creel"immobilized technology for our research."Fish-in-creel"route just has the same meanings with its literal meaning."fish-in-creel"immobilized technology means that the enzyme is added to encapsulation system after mesoporous molecular sieve is synthesized, as going fishing after netting-making. Mesoporous channel not only offers mass transfer channel but also presents reaction space. Comparing to"fish-in-net", the enzyme is loaded in cage-style reactor. The interaction between enzyme and silica-based matrix in"fish-in-creel"approach is the same as that in"fish-in-net"route."fish-in-creel"approach has an easy operation. Hence, it is reasonable and feasible in"fish-in-creel"to study the catalytic property of immobilized enzyme.Moreover,"fish-in-creel"approach can be performed under mild conditions. The processing of immobilization is reversible. Carriers can be repeatedly used. In a word, it is suitable in the industrial application.The major research of"Fish-in-creel"immobilization technology is that small fish is entrapped in big creel. It is easy for fish to access to creel and it can move around freely. Hence, it makes that it is very similar between molecular enzyme chemical reaction environment in molecular sieve carrier and natural enzyme reaction environment. And it benefits to rotation and change of enzyme molecules three-dimensional conformation in order to enhance the enzymatic activity. But it is easy to get out, since the interaction force between"fish"and"net"are relative weak. It is easy to be destroyed by micro-environment such as the high ionic strength and pH. The focus of our research is that how to seek the optimum working conditions in order to control the entry and exit of fish."Fish-in-creel"immobilization technology study three points: enzyme molecule, carrier, as well as micro-environment, in which immobilization enzyme activity is influenced by physical and chemical property. Therefore, based on physical and chemical properties of the carrier, matched enzyme is selected. In contrast, matched carrier is chosen according to the nature of immobilized enzyme.The first chapter of this thesis briefly introduces the basic knowledge of immobilized enzyme, the second chapter focuses on "Fish-in-Net" immobilization technology, as well as Fish-in-creel immobilization technology, the third chapter is the methods of experiment, the forth chapter is the results of experiment. In my story based on SBA-15 as a carrier for immobilization of trypsin, simple operation, mild conditions, and better preparation result. Mainly study the relations between the amount of immobilization enzyme and specific activity. (4.2), the situation of trypsin escapes from the carrier. ( 4.3), the activity of resistant acid-alkali is reversible, the optimum pH of immobilized trypsin and the best working conditions and so on, but also other indicators such as effects of temperature(4.4), viscosity(4.5). On the basis these works, the immobilization of trypsin will deeply studied for practice production.The experimental results show that direct proportion between the amount of immobilized enzyme and its activity are not obtained. When adequate (half saturated) trypsin is immobilized in the molecular sieve immobilize, giving full play to the efficiency of each enzyme molecule, reducing the self-dissolution of trypsin, and increasing the stability of enzyme. Small change of optimal pH (7.5) in immobilized trypsin occurs compared to that of free enzyme. This small difference may be related to nature of carriers and changes of enzyme conformational and the micro-environment. As the pH (3.0-10.6) increased, immobilized trypsin will gradually escape from molecular sieve through electrostatic adsorption. All of trypsin escaped from carriers at pH 10.6. Along with the escape, ceaseless changes of the reversibility of the activity of resistant acid-alkali occur. In production practice, we hope that immobilized trypsin has higher renatured capacity, lower leaching and denatured capacity. In this experiment, the optimal use of trypsin in industrial production is further analyzed, immobilized trypsin possess higher activity and lower leaching under the pH 7.5 conditions. Residual activity maintain more than 80% after be used three times in strong acid-alkali solution. In addition, on the basis experiment of temperature and viscosity, trypsin is widely applied in practice production. This immobilized method is simple, easy to operate, but also applicable to other enzyme immobilization.In this article, using mathematics modeling methods, the influence of pH to immobilization enzyme and the optimum working condition is discussed, and this method is simultaneously suitable for other enzyme immobilization. And it suggests that surface charge of enzyme is altered through genetic engineering method and that the charge of molecular sieve is transformed by chemistry modification method. It is provide theoretical support and technical guidance for the more researches of immobilized enzymes.
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