2009-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/705557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。然而木質纖維素的組成與結構極為複雜，其完全水解需仰賴多種酵素共同作用，僅利用單一酵素水解效果有限。因此，多功能纖維分解酵素的使用可加速木質纖維素的水解及減少酵素的使用量，對生產成本的降低有極大助益。自然界雖已存在諸多種類的雙功能或多功能纖維分解酵素，然而，以基因工程技術將個別高活性的纖維分解酵素基因接合而產製多功能的嵌合體酵素，為一較有效率的方式。本計畫擬利用基因工程技術，將瘤胃真菌的纖維素&#37238;、聚木糖&#37238;，以及乙醯聚木糖酯&#37238;基因進行接合，再利用酵母菌表達系統進行融合蛋白質的大量表達，以產製具有多功能纖維分解能力之嵌合體酵素。除探討嵌合體酵素的活性、酵素動力學、最適作用條件等基本特性外，並將利用發酵槽進行多功能嵌合體酵素之大量產製，再以本土農業廢棄物為基質，進行木質纖維素之水解試驗。此外，亦將評估嵌合體酵素應用在肉雞飼料之可行性。<br> Abstract: Agricultural residues represent large renewable resources for lignocelluloses bioconversion. Daily, the agri-industry generates significant amounts of by-products that are considered as polluting wastes to be eliminated. A great deal of research has been conducted on the utilization of these by-products in the biotechnology sector. As plant cell walls are structurally complex, bio-depolymerization of lignocellulosic biomass requires a large number of enzymes, many of which work synergistically to degrade complex polysaccharides. However, the requredment of multiple enzymes for lignocelluloses depolymerization is one of the obstacles to economical bioconversion as production of a large number of enzymes is energy and cost intensive. Challenges facing the cost-effective enzymatic saccharification of lignocellulosic biomass include the need for increased hydrolase activities and the reduction of the number of proteins required for the process. Engineering multidomain enzymes that are capable of catalyzing two or more reactions is a feasible way to reduce the number of enzymes in biomass conversion. In addition, multifunctional hydrolases are potentially synergistic in degrading complex substrates. Although the natural diversity of enzymes provides some candidates with evolved bi- or multi-functional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes. In this study, a multifunctional fibrolytic enzyme gene will be constructed by fusion the rumen fungal cellulose, xylanase, and acetylxylan esterase genes. The recombinant chimeric enzyme will be purified to investigate their characteristics, such as specific activity, substrate specificity, enzyme kinetics, and the optimal conditions for the highest enzyme activity. In addition, chimeric enzyme will also be produced by large scale fermentation and estimated its application in bioconversion of agricultural waste and broiler feed additive.纖維素&#37238聚木糖&#37238乙醯聚木糖酯&#37238嵌合體酵素Cellulasexylanaseacetylxylan esterasechimeric enzyme