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標題: Purification of Thermostable D-Hydantoinase from Transgenic Rice and Expression of Microbial Transglutaminase
作者: 楊明德
關鍵字: 生物技術, 農藝, 藥學;應用研究
Transglutaminases (TGase;glutaminyl-peptide: amine r-glutaminyltransferase, EC are enzymes that catalyze an acyl transfer reaction in which r-carboxyamide groups of protein-bound glutamine residues are the acyl donors. Intramolecular or Intermolecular cross-linking of certain proteins are generated by TGase through the formation of r-glutamyl-e-lysine side chain bridges between glutamine and lysine residues in proteins. TGase activity is found to be widely distributed in various cells and body fluids of mammals and thought to play important roles in cellular processes involving blood clotting, wound healing, and stabilization of the cell cytoskeletal network. The covalent cross-linking activity of TGase has also been used to improve the nutritive value and functional properties of food proteins. In recent years, studies on using TGase for food processing and the potential applications of TGase in medical treatment are increased. Thus, it would be worthwhile developing an efficient system for production of TGase. To facilitate mass production, TGases derived from microbial sources are subjected to be identified and characterized. By using DNA fragment from Streptoverticillium mobaraense TGase gene as probe, the gene corresponding to the Streptoverticillium kentuckense TGase was cloned and sequenced. In this study, a high-level expression system of the cloned TGase gene in E. coli will be constructed. Moreover, purification and characterization of the Streptoverticillium TGases will be conducted. The second part of this project will cooperate with professor L.J. Chen in National Chung Hsing University. We expect to establish a simple and fast method to isolate and purify the high-value thermostable D-hydantoinase from the transgenic rice plants provided by Dr. Chen's laboratory. The biochemical characteristics of the purified enzyme will also be examined.

麩胺酸醯基轉移? (Transglutaminase; EC 是種醯基轉移酵素,會造成蛋白質產生分子間或分子內的交叉架構 (cross-linking)。它的酵素活性可在細胞組織及體液中被偵測到,被認為和許多細胞的生理功能有關。由麩胺酸醯基轉移?所催化的聚合作用,在工業上亦被有效的應用於食品蛋白功能性的改善及營養價值的提昇,因此在食品加工及醫學上的應用深具潛力。目前此酵素具有高單價、市場潛力大的特色,而尋找非哺乳類的麩胺酸醯基轉移?來源,一直是業界努力的目標。我們在上一年度的計劃中,已從Streptoverticillium屬菌株中選殖出新的麩胺酸醯基轉移?酵素基因,有關基因的選殖與定序工作已經完成,我們希望能在大腸菌中大量表現出具有酵素活性蛋白,並進一步作酵素分離、純化及生化特性等評估工作,以提供作為高價位醫藥工業用酵素基因轉殖水稻的另一項來源。此外在第二部分的工作中,將與本校陳良築教授共同進行轉基因水稻耐熱性 D-hydantoinase (t-DHT) 酵素的純化與生化特性分析的工作。利用由陳教授所提供的轉tdht基因同質品系水稻,建立起簡單、快速的t-DHT酵素分離、純化方法,並利用純化的蛋白進行定量及生化特性分析的工作。
其他識別: 91農科-3.1.1-糧-Z4(6)
Appears in Collections:分子生物學研究所

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