Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum

• Published in: Journal of bioscience and bioengineering 2007-02, Vol.103 (2), p.174-178
• Main Authors: Yamada-Onodera, Keiko, Fukui, Masato, Tani, Yoshiki
• Format: Article
• Language: English
• Subjects: ( S)- N-benzyl-3-pyrrolidinol production; ALCOHOL DEHYDROGENASE; Alcohol Dehydrogenase - chemistry; Alcohol Dehydrogenase - isolation & purification; ALCOHOL DESHIDROGENASA; ALCOOL DESHYDROGENASE; AMMONIUM SULPHATE; Biological and medical sciences; Biotechnology; Chromatography, High Pressure Liquid; Dimerization; Electrophoresis, Polyacrylamide Gel; ENZIMAS; ENZYME; ENZYMES; Fatty Alcohols - chemistry; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Fungal Proteins - isolation & purification; GEOTRICHUM; Geotrichum - enzymology; Geotrichum capitatum; HPLC; N-benzyl-3-pyrrolidinol dehydrogenase; N-benzyl-3-pyrrolidinone reductase; optically pure ( S)- N-benzyl-3-pyrrolidinol; Oxidation-Reduction; Pyrroles - chemistry; Pyrroles - metabolism; Stereoisomerism; Substrate Specificity; SULFATE D'AMMONIUM; SULFATO DE AMONIO
• ISSN: 1389-1723; 1347-4421
• 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.