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ID 47684
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Purification and Characterization of Thermostable Amidase from Thermus sp.O-3-1
Author
Kobayashi, Fumiaki
Aomine, Hiroki
Mizunashi, Wataru
Yu, Fujio
Abstract
The gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally, as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.
Abstract Alternative
好熱性細菌Thermus sp.O-3-1 由来の耐熱性アミダーゼ遺伝子を大腸菌中にクローニングし,その塩基配列を決定した.ami 遺伝子は930 bp からなり,310アミノ酸をコードしていた.本酵素の分子量は33,089 Daであると予想された.Thermus sp.O-3-1 由来アミダーゼを大腸菌で生産させ,熱処理とDEAE-トヨパール650M陰イオン交換カラム等により精製した.ゲル濾過クロマトグラフィーとSDS-PAGE の結果から本酵素は分子質量33 kDa のサブユニット2分子からなるダイマー構造を有していることが明らかとなった.精製酵素の熱安定性は80℃まで,pH 安定性は7.0~10.0であり,安定性の 高い酵素であった.最適温度は90℃,最適 pH は9.0であ った.EDTA により活性が著しく阻害され,Co(2+)やNi(2+),Mn(2+)によって活性の回復,向上が見られたため,本酵素は金属酵素であることが示唆された.基質特異性の検討 の結果,L-Leu-pNA よりもD-Leu-pNA に対して高い活性を示したため,本酵素がD-アミノ酸基質に特異性を持つアミダーゼであることが判明した.本酵素は耐熱性を有するユニークなD-アミノ酸アミダーゼであり,今後産業利用が期待される.
Keywords
amidase
thermostable enzyme
Thermus
D-amino acid specific amidase
Note
原著論文 (Original paper)
Published Date
2012-02-01
Publication Title
岡山大学農学部学術報告
Publication Title Alternative
Scientific Reports of the Faculty of Agriculture Okayama University
Volume
volume101
Publisher
岡山大学農学部
Publisher Alternative
Faculty of Agriculture, Okayama University
Start Page
1
End Page
6
ISSN
2186-7755
Content Type
Departmental Bulletin Paper
language
日本語
File Version
publisher
Refereed
False
Eprints Journal Name
srfa