2005-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/671392摘要：工業上利用具有光學選擇性的天冬胺酸4-去羧&#37238;(Asd)，進行β去羧基反應生產丙胺酸，可 應用於食品添加劑及營養劑。比對天冬胺酸轉胺&#37238;進行三點突變使酵素轉為去羧&#37238;(J. Biol. Chem. 274, 31203-31208)，本研究將針對兩種酵素不同的胺基酸序列進行研究，同時利用DNA shuffling 改造Asd 具有轉胺&#37238;活性，並利用分子模擬解釋酵素分子中選擇反應路徑之重要胺基 酸。本計畫擬分三年執行: 第一年建構Pseudomonas sp.原態酵素之大腸桿菌表現株，試驗最佳 誘導條件及純化流程，並建構六個定點突變株，分析變異點對反應專一性之影響；第二年利用 重組Asd 與轉胺&#37238;基因片段來建構基因庫，並建立轉胺活性篩選法挑取轉胺活性增加之Asd， 將變異酵素純化並進行反應專一性之HPLC 分析。<br> Abstract: L-aspartate 4-decarboxylase (Asd) was used in the industrial production of L-alanine, as a food additive and a component of amino acid infusion, for its optical selectivity. In this study, amino acid residues that are different from Asd and aminotransferases according to the multiple sequence alignment will be investigated, since Graber et al. (J. Biol. Chem. 274, 31203-31208) had mutated three residues at the active site of aspartate aminotransferase and altered enzyme into an Asd. Enzyme engineering will be performed to enhance aminotransferase acitivity of Asd by DNA shuffling. Besides, molecular modeling of the Asd will be helpful in explaining the key amino acid residues that direct the reaction pathway. This project will be finished in three years. In the first year, a native Asd cloned from Pseudomonas sp. ATCC 19121 will be subcloned into an E. coli host, expressed, purified, and characterized. Furthermore, reaction specificity of the six site-directed mutations will be examined. In the second year, gene fragments from asd and aminotransferase will be assembled to construct the mutation library. Colonies with higher aminotransferase activity, screened by the oxaloacetate-Fast violet B coloring method, will be selected, and both enzyme activities will be assayed.天冬胺酸4-去羧&#37238轉胺&#37238DNA shufflingaspartate 4-decarboxylaseaminotransferaseDNA shuffling