Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597
Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597
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DOI：
10.1007/s00253-009-2329-5
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发表时间：
2010-03
影响因子：
5
通讯作者：
Y. Nakata;T. Fukae;Ryoji Kanamori;S. Kanamaru;T. Matsuda
中科院分区：
文献类型：
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作者：
Y. Nakata;T. Fukae;Ryoji Kanamori;S. Kanamaru;T. Matsuda
从土曲霉candidumNBRC 4597中分离得到具有高立体选择性和宽底物特异性的nadh依赖性酶还原苯乙酮衍生物，对其进行了纯化、表征并用于不对称合成。经硫酸铵分馏和一系列色谱等5步纯化，酶纯度约150倍，产率为5.6%。经凝胶过滤层析测定，活性酶的分子量为73 kDa， SDS-PAGE结果显示该亚基的分子量为36 kDa。这些结果表明该酶由一个36kda亚基的同二聚体组成。苯乙酮还原酶在50℃时活性最高，pH为5.5时活性最佳。酶在40℃时最稳定。金属离子Cu2+、Cd2+、Zn2+等金属离子对酶活性有较强的抑制作用。还原酶的vmax和表观km值分别为77.0µmol/min / mg / protein和0.296 mM / mg / protein。用肽段测序仪测定n端和内部氨基酸序列。将纯化后的酶用于苯乙酮的不对称还原，得到相应的ee含量为99%的(S)-醇。
NADH-dependent enzyme reducing acetophenone derivatives with high stereoselectivities and wide substrate specificities fromGeotrichum candidumNBRC 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. TheVmaxand the apparentKmvalue 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.