論文 - 詳細
| RRC ID | 5016 |
|---|---|
| 著者 | Yamada-Onodera K, Fukui M, Tani Y. |
| タイトル | Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum. |
| ジャーナル | J Biosci Bioeng |
| Abstract |
(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds. |
| 巻・号 | 103(2) |
| ページ | 174-8 |
| 公開日 | 2007-2-1 |
| DOI | 10.1263/jbb.103.174 |
| PII | S1389-1723(07)70043-9 |
| PMID | 17368401 |
| MeSH | Alcohol Dehydrogenase / chemistry* Alcohol Dehydrogenase / isolation & purification Chromatography, High Pressure Liquid Dimerization Electrophoresis, Polyacrylamide Gel Fatty Alcohols / chemistry Fungal Proteins / chemistry* Fungal Proteins / isolation & purification Geotrichum / enzymology* Oxidation-Reduction Pyrroles / chemistry* Pyrroles / metabolism* Stereoisomerism Substrate Specificity |
| IF | 2.366 |
| 引用数 | 20 |
| WOS 分野 | FOOD SCIENCE & TECHNOLOGY BIOTECHNOLOGY & APPLIED MICROBIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 6 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM3908 |