Purification and Characterization of Alcohol Dehydrogenase Reducing N-Benzyl-3-Pyrrolidinone from Geotrichum capitatum(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)
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概要
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(S)-N-Benzyl-3-pyrroIidinol 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 78kDa. 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-S-pyrrolidinol oxidation were 0.13 and 8.47mM, 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.
- 社団法人日本生物工学会の論文
- 2007-02-25
著者
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Yamada-Onodera Keiko
Graduate School of Biological Sciences, Nara Institute of Science and Technology
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Tani Yoshiki
Graduate School of Biological Sciences, Nara Institute of Science and Technology
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Tani Yoshiki
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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Yamada-onodera Keiko
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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Fukui Masato
Graduate School of Biological Sciences, Nara Institute of Science and Technology
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Fukui Masato
Graduate School Of Biological Sciences Nara Institute Of Science And Technology
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Tani Yoshiki
Graduate School Of Biological Sciences Advanced Institute Of Science And Technology
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Yamada-onodera Keiko
Graduate School Of Biological Sciences Nara Inst. Of Sci. And Technol.
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