Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan.
Appl Microbiol Biotechnol. 2010 Mar;86(2):625-31. doi: 10.1007/s00253-009-2329-5. Epub 2009 Nov 12.
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.
从 NBRC4597 中分离、纯化、表征并用于不对称合成的 NADH 依赖性酶，可高立体选择性和广泛的底物特异性还原苯乙酮衍生物。通过包括硫酸铵分级和一系列色谱在内的五步纯化，该酶的纯度提高了约 150 倍，产率为 5.6%。凝胶过滤色谱法测定该酶的活性酶具有 73 kDa 的分子量，SDS-PAGE 结果表明亚基的分子量为 36 kDa。这些结果表明该酶由 36 kDa 亚基的同源二聚体组成。苯乙酮还原酶在 50°C 时表现出最高活性，最佳 pH 值为 5.5。该酶在 40°C 时最稳定。没有金属离子能显著激活该酶，而 Cu2+、Cd2+和 Zn2+等金属离子强烈抑制该酶的活性。该还原酶的 Vmax 和表观 Km 值分别为 77.0 μmol/min/mg 蛋白和 0.296 mM 对苯乙酮。通过肽测序仪确定了 N 端和内部氨基酸序列。此外，还使用纯化的酶对苯乙酮进行了不对称还原，形成了相应的 (S)-醇，ee 值为 99%。
