Halohydrin dehalogenases (HHDHs) are valuable biocatalysts involved in the synthesis of β-substituted alcohols via their nucleophile-mediated ring-opening activity. To use directed evolution to unleash the latent potential of HHDHs for the synthesis of β-substituted alcohols, we report a high-throughput assay for screening HHDHs mutagenesis libraries. The assay is performed in a 96-well microtiter plate format using a cell-free extract or whole-cells in the presence of the desired nucleophile. The developed method relies upon the color change of bromothymol blue, due to the pH change caused by HHDH-catalyzed ring-opening of the epoxide substrate. The assay was validated using gas chromatography and subsequently applied to high-throughput screening of halohydrin dehalogenase HheC mutagenesis library. Active mutants were found for the tested substrates. Due to its simplicity and flexibility towards the use of nucleophiles and epoxides, the method is an attractive alternative to the existing assays for HHDH epoxide ring-opening reaction and could be helpful in the rapid discovery of industrial biocatalysts.

中文翻译：

---

一种高通量筛选测定法，用于针对环氧化物开环反应的卤代醇脱卤酶突变体的定向进化引导发现。

卤代醇脱卤酶（HHDHs）是有价值的生物催化剂，其通过亲核试剂介导的开环活性参与β-取代醇的合成。为了使用定向进化来释放HHDHs潜在的合成β-取代醇的能力，我们报道了用于筛选HHDHs诱变文库的高通量检测方法。在所需亲核试剂存在下，使用无细胞提取物或全细胞以96孔微量滴定板形式进行测定。由于由HHDH催化的环氧底物开环引起的pH值变化，所开发的方法依赖于溴百里酚蓝的颜色变化。使用气相色谱法验证了该测定，随后将其应用于卤代醇脱卤酶HheC诱变文库的高通量筛选。发现了用于测试底物的活性突变体。由于其使用亲核试剂和环氧化物的简便性和灵活性，因此该方法是HHDH环氧化物开环反应的现有测定方法的一种有吸引力的替代方法，可有助于快速发现工业生物催化剂。