Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.
Mol Biotechnol. 2013 Oct;55(2):143-9. doi: 10.1007/s12033-013-9666-4.
Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.
由于酶具有显著的催化特性，例如其广泛的底物特异性和多种化学、立体和区域选择性反应，用生物催化替代化学步骤变得越来越有趣。本研究介绍了嗜盐古菌 Halobacterium sp. NRC-1（HsADH2）中的醇脱氢酶（ADH）的特性。HsADH2 的六组氨酸标记重组版本（His-HsADH2）在 Haloferax volcanii 中异源过表达。该酶通过固定化 Ni 亲和层析一步纯化。His-HsADH2 是嗜盐的，略带嗜热，在 4 M KCl 下最适乙醇氧化活性约为 60°C 和 pH 10.0。该酶非常稳定，在 30 天后保留 80%的活性。His-HsADH2 优先使用 NADP(H)，但有趣的是对 NADH 保留 60%的活性。该酶显示出广泛的底物特异性，最大活性获得在 1-丙醇。该酶还接受仲醇，如 2-丁醇，甚至 1-苯乙醇。在还原反应中，优化了 His-HsADH2 用于乙醛的工作条件，发现最佳条件为 4 M KCl 和 pH 6.0。His-HsADH2 表现出内在的有机溶剂耐受性，这对生物技术应用非常重要。
