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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  Agrobacterium radiobacter N-carbamoyl-D-amino acid amiidohydrolase之特性分析及改良

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/92789

標題: Agrobacterium radiobacter N-carbamoyl-D-amino acid amiidohydrolase之特性分析及改良
Characterization and Protein engineering ofN-carbamoyl-D-amino acid amiidohydrolase of Agrobacterium radiobacter
作者: 徐銓龍
Hsu, Chuan-Long
Contributors: 許文輝
Hsu, Wen-Hwei
國立中興大學
關鍵字: 分離;蛋白質電泳
日期: 1998
Issue Date: 2012-08-31 12:02:50 (UTC+8)
Publisher: 分子生物研究所
摘要: N-carbamoyl-D-amino acid amidohydrolase (Nca)能將N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG)水解成為D-hydroxyphenylglycine (D-p-HPG),是一種相當重要的工業用酵素。本研究由Aguobacterium radiobacter選殖nca基因,並使其在Escherichia coli菌體內大量表現。經分離、純化及蛋白質電泳分析後,發現Nca的分子量大約為36kDa。
Nca經50℃熱處理5個小時後,會完全喪失其酵素活性。以0.4mMH2O2處理Nca後,會逐漸喪失其酵素活性。除了Cu2+離子對Nca活性有嚴重的抑制之外,其餘金屬離子對Nca活性並無明顯的提升或抑制的現象。Nca的活性也不會被終產物NH4+所抑制。
利用質子光譜分析氧化態及原態Nca分子量間的差異,發現兩者相差27Da,由此推測methionine可能是被氧化的氨基酸殘基,為了鑑定被氧化的methionine殘基在Nca之位置,將Nca上所有的methionine殘基以定點突變法置換成為leucine殘基,再測定變異酵素的抗氧化性,發現以10mMH2O2處理後,Met-239/Met-244變異Nca之比活性維持7.0U/mg,顯示不受H2O2影響,推測Met-239或Met-244可能是Nca被氧化的氨基酸殘基。
為了提升Nca的酵素活性,以利於D-p-HPG的生產,本研究利用PCR任意點突變配合多倍化質體篩選法,突變nca基因,由1,000株菌中篩選到4株突變株,其粗酵素液活性較野生株高出1.5倍。惟經繼代培養,再純化回收酵素,測定其比活性的結果,和野生株比較並無明顯的差異。
為了推測那些氨基酸殘基參與Nca酵素的催化反應,利用電腦軟體GCG比對A.radiobacter及Pseudomonas sp.的Nca氨基酸序列,發現有9個aspartate及4個histidine殘基位於保留區當中。以定點突變法將上述13個氨基酸殘基分別置換成為alanine,並分析該突變點對酵素活性的影響。發現Asp-209-Ala及His-129-Ala完全失去活性。將Asp-209置換成glutamate殘基時,比活性則可恢復30%,由此推斷Asp-209殘基的carboxy1 group可能在Nca催化區內,扮演重要角色。以0.5mM diethylpyrocarbonate處理後,Nca即喪失其酵素活性,顯示histidine殘基在Nca的催化區內相當重要。將His-129突變為arginine及asparagine,發現突變酵失去活性,推測His-129在Nca酵素催化區內,可能並非參與氫鍵的形成而是扮演質子轉移的功能。
The N-carbamoyl-D-amino acid amidohydrolase (Nca) is an important enzyme used in the bioconversion of N-carbamoyl-D-p-hydroxyphenylglycine (Nca-HPG-) to D-p-hydroxyphenylglycine (D-p-HPG). The nca gene of Agrobacterium radiobacter was cloned and expressed in Escherichia coli. The Nca is a homodimeric enzyme with subunit molecular mass of 36 kDa.
Enzymatic activity of Nca is inactivated after 5 h of incubation at 50℃. To examine the oxidation stability of Nca, the enzyme was incubated at 37℃ in the presence of different concentrations of H2O2. Mass spectrum revealed that artificial oxidation of Nca with 0.4 mM H2O2 was too harsh to discriminate oxidized residues. Molecular mass of Nca inactivatede under natural oxidation was increased by 27 Da compared to that of native enzyme. Therefore, the methionine residues of Nca seem to be oxidized by oxygen. To verify whether methionine residues were oxidized, all methionine residues in Nca were substituted by leucine via site-directed mutagenesis. The results revealed that mutant Met-239-Leu/Met-244-Leu retained its activity after H2O2 treatment, indicating that Met-239 or Met-244 might be involved in enzyme oxidation.
To improve the Nca activity, PCR-based random mutation coupled plasmid multimerization was applied to nca gene evolution. Of one thousand mutants, the Nca activities of four mutants were increased by 1.5-fold compared to that of the parent strain, but specific activity remained unchanged.
Amino acid sequence alignment of A. radiobacter Nca and Pseudomonas sp. Nca displayed 58.6% identity. Based on this identity, a number of aspartic acid and histidine residues in conserved regions of A. radiobacter Nca were selected for site-directed mutagenesis to evalute the effect of these amino acid residues on Nca activity. Nca mutants, Asp-209-Ala and His-129-Ala, lost their activities completely, suggesting that Asp-209 and His-129 residues might be important in catalysis. The Asp-209-Glu mutant retained 30% activity, indicating that carboxyl group of Asp-209 participated in catatlysis. Substitution of His-129 by asparagine or arginine abolished enzyme activity completely, suggesting that His-129 might be involved in proton transfer, but not in hydrogen bonding.
Appears in Collections:[依資料類型分類] 碩博士論文

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