Bakonyi Daniel, Hummel Werner
Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, Germany.
Institute of Molecular Enzyme Technology, Heinrich Heine University of Düsseldorf, Research Centre Jülich, Wilhelm-Johnen-Straße, 52426 Jülich, Germany.
Enzyme Microb Technol. 2017 Apr;99:16-24. doi: 10.1016/j.enzmictec.2016.12.006. Epub 2016 Dec 27.
A gene encoding a novel 7α-specific NADP-dependent hydroxysteroid dehydrogenase from Clostridium difficile was cloned and heterologously expressed in Escherichia coli. The enzyme was purified using an N-terminal hexa-his-tag and biochemically characterized. The optimum temperature is at 60°C, but the enzyme is inactivated at this temperature with a half-life time of 5min. Contrary to other known 7α-HSDHs, for example from Clostridium sardiniense or E. coli, the enzyme from C. difficile does not display a substrate inhibition. In order to demonstrate the applicability of this enzyme, a small-scale biotransformation of the bile acid chenodeoxycholic acid (CDCA) into 7-ketolithocholic acid (7-KLCA) was carried out with simultaneous regeneration of NADP using an NADPH oxidase that resulted in a complete conversion (<99%). Furthermore, by a structure-based site-directed mutagenesis, cofactor specificity of the 7α-HSDH from Clostridium difficile was altered to accept NAD(H). This mutant was biochemically characterized and compared to the wild-type.
编码来自艰难梭菌的一种新型7α特异性NADP依赖性羟类固醇脱氢酶的基因被克隆，并在大肠杆菌中进行了异源表达。使用N端六组氨酸标签对该酶进行纯化，并对其进行生化特性分析。最适温度为60°C，但该酶在此温度下会失活，半衰期为5分钟。与其他已知的7α-HSDHs（例如来自沙丁鱼梭菌或大肠杆菌的）不同，艰难梭菌的这种酶没有表现出底物抑制作用。为了证明这种酶的适用性，利用NADPH氧化酶进行了胆酸鹅去氧胆酸（CDCA）向7-酮石胆酸（7-KLCA）的小规模生物转化，同时再生NADP，转化率达到了完全转化（<99%）。此外，通过基于结构的定点诱变，改变了艰难梭菌7α-HSDH的辅因子特异性，使其能够接受NAD(H)。对该突变体进行了生化特性分析，并与野生型进行了比较。
