A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.
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 k(cat) values, 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 k(cat) value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, respectively, although it exhibits higher K(m) values 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.
Tang
Lixia
L
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. lixiatang@uestc.edu.cn
Li
Yang
Y
Wang
Xiong
X
D002021:Buffers; D003124:Colorimetry / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis / Q000235:genetics*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010637:Phenolsulfonphthalein / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D013057:Spectrum Analysis
D002021:Buffers; D007202:Indicators and Reagents; D050505:Mutant Proteins; D006867:Hydrolases; C430848:halohydrin dehalogenase; D010637:Phenolsulfonphthalein