School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China.
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China; Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China.
J Biotechnol. 2020 Mar 10;311:19-24. doi: 10.1016/j.jbiotec.2020.02.007. Epub 2020 Feb 14.
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.
卤代醇脱卤酶（HHDHs）是一种有价值的生物催化剂，通过亲核介导的开环活性参与β-取代醇的合成。为了利用定向进化释放 HHDHs 在β-取代醇合成中的潜在潜力，我们报告了一种用于筛选 HHDHs 突变文库的高通量测定法。该测定法在存在所需亲核试剂的情况下，以无细胞提取物或整个细胞的形式在 96 孔微量滴定板格式中进行。该方法依赖于溴百里酚蓝的颜色变化，这是由于 HHDH 催化环氧化物底物开环引起的 pH 变化。该测定法使用气相色谱法进行了验证，随后应用于卤代醇脱卤酶 HheC 突变文库的高通量筛选。针对测试的底物发现了活性突变体。由于该方法具有简单性和对亲核试剂和环氧化物的灵活性，因此是现有 HHDH 环氧化物开环反应测定法的一种有吸引力的替代方法，并且可能有助于快速发现工业生物催化剂。
