2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/37773L-Amino acids, used as a precursor of many pharmaceuticals which function as ACE-inhibitors, protease inhibitors and neuronal receptor ligands, are made from N-carbamoyl-L-amino acids produced by hydrolysis of the corresponding 5-substituted hydantions by the hydrolyzing enzyme L-hydantoinase, or from N-acetyl-L-amino acids hydrolyzed by the enzyme L-aminoacylase. An efficient synthesis of chiral L-amino acids used industrially is enantio-selective hydrogenation. Instead of the chemical process, an enzymatic process involving either L-N-carbamoylase(Lnca) or L-aminoacylase(Laa)has several advantages.The distribution of Laa activity in microorganisms was investigated. High activity was found in various bacteria belonging to genera Alcaligenes denitrificans, Bacillus stearothermophilus, Alcaligenes faecalis, and Alcaligenes xylosoxydans. The laa gene had been cloned and identified from B. stearothermophilus. The Laa is a homotetrameric enzyme with subunit molecular mass of 42 kDa. Enzymatic activity of Laa is inactivated without the additon of metal ions, however, be progressively restored by increasing Co2+ or Mn2+ concentration. The deacetylating capacity of Laa varies considerably depending on the nature of the amino acid residue in the substrate. The Laa hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently.The Laa enzyme has been studied by several groups, but only the gene encoding L-amino acylase from B. stearothermophilus has been cloned and expressed. However, the amino acid and DNA sequences of L-aminoacylase from B. stearothermophilus is already published and patented. To solve above problems, We have cloned the upper portion of laa gene from Aspergillus oryzae and also identified four L-aminoacylase-producing bacteria, thermophilic Bacillus caldovelox, Bacillus caldolyticus, Basillus licheniformis and Bacillus sp. TS-23. The research goals of this project are followings.1. Cloning and characterization of laa genes from A. oryzae B. caldovelox, B. caldolyticus, B. licheniformis and Bacillus sp. TS-23.2. Characterization of A. oryzae L-aminoacylase.3. Preparation of laa DNA for gene trausfer into rice.L型胺基酸是用來合成ACE-inhibitor,protease inhibitor及neuronal receptor ligands的原料,它可由5-substituted hydantoins經L型hydantoinase水解而成N-carbamoyl-L-amino acid再一次水解而成.或由N-acetyl-L-amino acid經L-aminoacylase(Laa)水解而成.在工業上有效地將DL-amino acid選擇性轉成L-amino acid的方法稱enantionselective hydrogenation,但它對L型胺基酸產量低摻雜些許多D型胺基酸,而且使用大量化學溶劑對環境造成非常大的衝擊,取而代之的是酵素L-N-aminoacylase(Lnca)或L-aminoacylase(Laa)水解流程,它們都可解決上述的問題.有很多研究團體在探討Laa生產菌,發現Alcaligens denitrificans Bacillus stearothermophilus, Alcaligens faecalis和Alcaligens xylosoxydans對Laa的生產能力較強,其中B. stearothermophilus的laa基因已被選殖及定序.B. stearothermophilus的laa基因選殖入E.coli中表現,發現Laa是homotetramer單體分子量為42kDa.在沒有金屬離子存在下,此Laa酵素活性盡失,若增加Co2+或Mn2+濃度,則Laa活性會再顯現.去乙醯基的能力強弱決定在於胺基酸殘基,發現此Laa最喜好水解環狀胺基酸殘基的N-乙醯衍生物.截至目前為止,僅有B. stearothermophilus的laa基因被選殖及定序.然而其DNA sequence及amino acid sequence已發表,使用時可能會抵觸國外專利.我們最近選殖到嗜熱性(最適生長溫度55 ℃) Aspergillus oryzae的laa基因前三分之一片段,正在進行完整laa基因選殖並進行DNA定序之中,也發現嗜高溫(最適生長溫度55 ℃) Bacillus caldovelox,Bacillus caldolyticus Bacillus sp. TS-23和嗜熱菌Bacillus licheniformis(最適生長溫度45 ℃)具有Laa活性,是選殖耐熱型Laa基因的好材料,突破國外專利保護的機會,值得繼續投入研發工作.這些研發工作,在基礎及應用科技領域,均有相當大的價值.本計畫將進行下列工作: 1. 選殖A. oryzae, B. caldovelox, B. caldolyticus, B. licheniformis及Bacillus sp. Ts-23的laa基因,完成DNA定序及基因結構分析工作. 2. 完成A. oryzae Laa的生化性質分析工作,特別是了解基耐熱性及對N-acetyl-L-HPA的Km及Kcat. 3. 將laa基因給與興大進行水稻基因轉殖.Project