2014-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/699849摘要：木質纖維素為地球上產量最豐富的再生資源，廣泛存在於農林作物中，可用來做為糧食、飼料、造紙、醫藥之原料。木質纖維素的組成與結構極為複雜，組成中包含了纖維素、半纖維素，以及木質素等，故其完全水解需仰賴多種酵素共同作用。此外，纖維素的結構常排列堆疊成緊密的結晶性纖維素，而大多數纖維素&#37238;對結晶性纖維素活性不高，若能提高纖維素&#37238;對結晶性纖維素的水解能力，將可加速木質纖維素的水解。本計畫為期二年，第一年將針對具有高結晶性纖維素水解活性的CbhYW23-2進行改造。將以理論設計法與定向演化法進行CbhYW23-2的基因突變，以提高酵素的活性與熱穩定性。並將比較CbhYW23-2及其突變酵素的比活性、酵素動力學、受質專一性，以及最適作用條件等基本特性。此外，亦將以X射線結晶分析法進行CbhYW23-2突變酵素的晶體結構研究，以瞭解突變所致之蛋白質結構改變與酵素特性間的相關性。第二年將利用適合工業化大量產製重組蛋白質的嗜甲醇酵母菌Pichia pastoris進行CbhYW23-2及其突變酵素之大量表達，並評估其做為飼料添加劑與應用於產製纖維素酒精之可行性。<br> Abstract: The agroindustry generates significant amounts of by-products that are considered as polluting wastes to be eliminated. These by-products include a great amount of lignocellulose, which are one of the most abundant renewable resources on earth. Therefore, a great deal of research has been conducted on the utilization of the lignocellulose in the biotechnology sector. As lignocellulose is composed of cellulose, hemicellulose, and lignin, bio-depolymerization of lignocellulosic biomass requires a large number of enzymes, many of which work synergistically. In addition, an important feature of cellulose is its crystalline structure. However, most cellulase cannot hydrolyze crystal cellulose efficiently. The efficiency of cellulose hydrolysis may be greatly improved if the enzyme activity of cellulase toward crystal cellulose is increased. This project will be implemented for two years. In the first year, CbhYW23-2, a cellulase shows high crystal cellulose hydrolyzed activity, will be mutated by using rational design or directed evolution strategy to improve its activity and/or thermal stability. The specific activity, enzymatic kinetic parameters, substrate specificity, and optimal reaction conditions of the mutant CbhYW23-2 will also be determined. In addition, the structure of mutant CbhYW23-2 will be studied by using X-ray crystallography in order to realize the relationship between protein structure and enzyme characteristics. In the second year, CbhYW23-2 and its mutants will be expressed by methylotrophic yeast Pichia pastoris. The recombinant enzymes will be produced by large-scale fermentation and their potentials to be applied in broiler feed additives and cellulosic ethanol production will be studied.纖維素&#37238理論設計法定向演化法X射線結晶分析法cellulaserational designdirected evolutionX-ray crystallography