Yamada-Onodera Keiko, Fukui Masato, Tani Yoshiki
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan.
J Biosci Bioeng. 2007 Feb;103(2):174-8. doi: 10.1263/jbb.103.174.
(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.
(S)-N-苄基-3-吡咯烷醇作为一种手性结构单元，在药物合成中被广泛应用。我们利用头状地霉JCM 3908的静息细胞反应，在28小时内从相应的酮N-苄基-3-吡咯烷酮中制备出了30 mM的(S)-N-苄基-3-吡咯烷醇（对映体过量>99.9%），产率超过99.9%。通过硫酸铵分级沉淀以及一系列DEAE-琼脂糖凝胶、丁基-琼脂糖凝胶、Superdex 200和羟基磷灰石柱色谱法，将头状地霉JCM 3908中依赖NAD(+)的将N-苄基-3-吡咯烷酮还原的醇脱氢酶纯化至均一。SDS-PAGE和HPLC结果表明该酶是一种分子量为78 kDa的二聚体。纯化后的酶能从N-苄基-3-吡咯烷酮生成(S)-N-苄基-3-吡咯烷醇（对映体过量>99.9%）。该酶还原2,3-丁二酮、2-己酮、环己酮、丙醛、正丁醛、正己醛、正辛醛、正戊醛和苄基丙酮的效率高于还原N-苄基-3-吡咯烷酮。未检测到对N-苄基-2-吡咯烷酮或2-吡咯烷酮的活性。在所采用的测定条件下，未检测到对(R)-N-苄基-3-吡咯烷醇的活性。该酶对2-丙醇、2-丁醇、2-戊醇、2-己醇、3-己醇和1-苯基-2-丙醇的氧化活性高于对(S)-N-苄基-3-吡咯烷醇。N-苄基-3-吡咯烷酮还原和(S)-N-苄基-3-吡咯烷醇氧化的K(m)值分别为0.13和8.47 mM。据我们所知，这是首次从真核生物中纯化出N-苄基-3-吡咯烷醇/N-苄基-3-吡咯烷酮氧化还原酶；此外，这也是微生物中(S)-N-苄基-3-吡咯烷醇脱氢酶活性的首次报道。该酶表现出与已知原核N-苄基-3-吡咯烷酮还原酶不同的特性。这种酶对于手性化合物的生产将非常有用。
Zotero 插件