A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.
A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.
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DOI：
10.1016/j.jbiotec.2010.04.002
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发表时间：
2010-06
影响因子：
4.1
通讯作者：
Lixia Tang;Yang Li;Xiong Wang
中科院分区：
文献类型：
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作者：
Lixia Tang;Yang Li;Xiong Wang
在这里，我们报告了一个高通量的pH指示剂为基础的测定卤代醇脱卤酶（HheC）的活性。该测定依赖于在560 nm处的吸光度变化和在弱缓冲体系中由于酶催化的闭环反应释放质子而导致的酚红的视觉颜色变化。该测定可以使用全细胞以微孔板形式进行，使得该测定简单且稳健。因此，它适合于文库筛选。使用两种先前研究的HheC变体D80 N和W249 F进一步验证了该测定，这两种变体对1，3-二氯-2-丙醇的kcat值分别比野生型HheC低200倍和高2倍。此外，使用开发的测定法筛选HheC的饱和诱变文库，以确定其有效催化1，3-二氯-2-丙醇转化的能力。经过500个菌落的筛选，鉴定出一个突变体W139 C，并进一步纯化和表征。动力学分析表明，所得到的突变体显示2-和5-倍的kcat值分别向1，3-DCP和（R，S）-对硝基-2-溴-1-苯基乙醇，虽然它表现出更高的Km值比野生型酶。本文所述的方法代表了考虑到卤代醇脱卤酶突变体的高通量筛选的需要的有用工具。
Here we have reported a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC). The assay relies upon the absorbance change at 560nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher kcatvalues, respectively, toward 1,3-dichloro-2-propanol than the wild-type HheC. In addition, a saturation mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-dichloro-2-propanol. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic analysis indicates that the resulting mutant shows 2- and 5-fold improvement in kcatvalue toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, respectively, although it exhibits higher Kmvalues than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants.