2004-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/683598摘要：N-acylamino acid racemase (NAAAR) 為一可針對N-端醯基化衍生胺基酸行消旋反應，但是對一般胺基酸則無消旋反應的消旋酵素，其可被應用於生產具光學活性之胺基酸。工業上利用D,L-N-acylamino acid 為基質生產D型胺基酸，單獨使用D-aminoacylase則基質轉換率最高僅能達到50 %，若協同使用NAAAR固定化生產D型胺基酸，則基質轉換率可提高至100 %。此外反應產物 D-胺基酸由於不會被 NAAAR消旋，故無須分離而可留存於反應液中，可以簡化生產 D-胺基酸之產程。本研究室已由Amycolatopsis azurea CCRC13413取得N-acylamino acid racemase 之基因，並建構於E.coli 表現系統，完成特性分析之研究。然而，在相關的研究中，N-acylamino acid racemase 於菌體內所扮演的生理功能尚不清楚，且由於目前所分離出之NAAAR酵素活性偏低，尚無法合適的應用於工業生產用途。為此，本計畫擬分別探究NAAAR於Amycolatopsis azurea CCRC13413所可能扮演的生<br> Abstract: NAAAR is an exceptional enzyme that can catalyzes the racemization of optically active N-acetyl amino acids but does not act on optically active amino acids. Optically active amino acid could be produced by immobilized NAAAR and D- or L-aminoacylase from N-acetyl-DL-amino acid. The final yield of amino acid from both enzymes will be increased to more than 99%, which is higher than 50% yield from D-aminoacylase alone. On our study, the N-acylamino acid racemase has been cloned and expressed the protein in E. coli, and the enzyme’s characterizations have been done. In all relative study, the real physiology role of the NAAAR in bacteria is still an open question. And, the activity of NAAAR from Amycolatopsis azurea or the other sources is not good enough for the application in industry. To solve the above questions, this study will confer the possible role of the N-acylamino acid arcemase in Amycolatopsis azurea and trying to use protein engineering to improve the enzyme activity and stability. The goal of the future is described as following: (1) Cloning the upstream and the down stream DNA of aaar gene from Amycolatopsis azurea. to analysis these genes function, and doing the phylogenetic analysis; (2) Using the methods of DNA shuffling and protein engineering to improve the enzyme stability and activity; (3) Developing an optima condition to produce the high specific activity of NAAAR.基因選殖基因表現蛋白質工程光學專一性N-acylamino acid racemasegene cloninggene expressionprotein engineeringoptical specificity.