Abstract
NADH-dependent enzyme reducing acetophenone derivatives with high stereoselectivities and wide substrate specificities from Geotrichum candidum NBRC 4597 was isolated, purified, characterized, and used for asymmetric synthesis. Through five-step purification including ammonium sulfate fractionation and a series of chromatographies, the enzyme was purified about 150-fold with a yield of 5.6%. The active enzyme has a molecular mass of 73 kDa determined by gel filtration chromatography, and the SDS-PAGE result reveals that the molecular size of the subunit is 36 kDa. These results indicate that the enzyme consists of a homodimer of a 36 kDa subunit. The acetophenone reductase exhibited the highest activity at 50°C and optimal pH at 5.5. The enzyme was the most stable at 40°C. No metal ions considerably activated the enzyme, and such metal ions as Cu2+, Cd2+, and Zn2+ strongly inhibited the activity of the enzyme. The V max and the apparent K m value of the reductase were 77.0 µmol/min per milligram of protein and 0.296 mM for acetophenone, respectively. The N-terminal and internal amino acid sequences were determined by peptide sequencer. Furthermore, the purified enzyme was used for asymmetric reduction of acetophenone, resulting in the formation of corresponding (S)-alcohol with 99% ee.
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Acknowledgements
We thank the Center for Advanced Materials Analysis, Tokyo Institute of Technology, Japan, for analysis of the amino acid sequence. The authors greatly appreciate the advice and technical support given by Professor Kaoru Nakamura at Kyoto University, Professor Tadao Harada, Mako Kuwahara, and Daisuke Taneike at Ryukoku University, and Professor Fumio Arisaka at the Tokyo Institute of Technology.
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Nakata, Y., Fukae, T., Kanamori, R. et al. Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597. Appl Microbiol Biotechnol 86, 625–631 (2010). https://doi.org/10.1007/s00253-009-2329-5
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DOI: https://doi.org/10.1007/s00253-009-2329-5