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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries
Lixia Tang1, Yang Li, Xiong Wang
1School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. lixiatang@uestc.edu.cn
Journal of Biotechnology
|April 20, 2010
Summary
A new pH indicator assay enables high-throughput screening of halohydrin dehalogenase (HheC) activity. This simple, whole-cell assay identifies improved enzyme variants for industrial applications.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Biochemical Assays
Background:
- Halohydrin dehalogenases (HheC) are enzymes with significant industrial potential.
- Existing methods for screening HheC activity are often low-throughput.
- Developing efficient screening tools is crucial for enzyme engineering.
Purpose of the Study:
- To develop a high-throughput, pH indicator-based assay for measuring halohydrin dehalogenase (HheC) activity.
- To validate the assay's robustness and suitability for library screening.
- To identify novel HheC mutants with improved catalytic efficiency.
Main Methods:
- A pH indicator-based assay using phenol red and absorbance changes at 560nm.
- Microplate format utilizing whole cells for simplicity and robustness.
- Saturation mutagenesis library screening of HheC for improved 1,3-dichloro-2-propanol conversion.
Main Results:
- The assay accurately reflects HheC activity through proton release and color change.
- Validated with known HheC variants (D80N, W249F) showing altered k(cat) values.
- Identified a novel HheC mutant (W139C) with enhanced k(cat) for 1,3-DCP and other substrates.
Conclusions:
- The developed assay is a simple, robust, and high-throughput tool for screening HheC activity.
- Facilitates the discovery of enzyme variants with improved catalytic properties.
- Represents a valuable method for advancing halohydrin dehalogenase engineering.
