Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597.
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 degrees C and optimal pH at 5.5. The enzyme was the most stable at 40 degrees 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 Vmax and the apparent Km value of the reductase were 77.0 micromol/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.
Nakata
Yasuo
Y
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Fukae
Takuro
T
Kanamori
Ryoji
R
Kanamaru
Shuji
S
Matsuda
Tomoko
T
D000098:Acetophenones / Q000378:metabolism*; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005848:Geotrichum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D021122:Protein Subunits; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D000098:Acetophenones; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008670:Metals; D021122:Protein Subunits; D010088:Oxidoreductases; C038699:acetophenone