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Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites
Su Jin Kim1, Jeong Chan Joo, Bong Keun Song
1Interdisciplinary Program of Bioengineering, School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, Korea; Korea Research Institute of Chemical Technology, Daejeon, 305-600, Korea.
Biotechnology and Bioengineering
|October 23, 2014
Summary
Protein engineering enhances peroxidase stability. Mutating vulnerable phenylalanine residues in horseradish peroxidase isozyme C (HRPC) significantly improves its resistance to inactivation by phenoxyl radicals during oxidative polymerization.
Area of Science:
- Biocatalysis and Protein Engineering
- Enzyme Stability and Industrial Biotechnology
Background:
- Peroxidases are valuable industrial biocatalysts, particularly for phenolic compound polymerization.
- Industrial use of peroxidases is hindered by rapid inactivation from phenoxyl radicals during polymerization.
Purpose of the Study:
- To develop a protein engineering strategy to enhance the radical stability of horseradish peroxidase isozyme C (HRPC).
- To identify and engineer phenylalanine residues susceptible to phenoxyl radical modification.
Main Methods:
- Mass spectrometry to identify radical-vulnerable phenylalanine residues.
- Site-directed mutagenesis to create single and multiple phenylalanine-to-alanine mutants.
- Biochemical characterization including UV-Vis and CD spectroscopy to assess enzyme activity and structure.
Main Results:
- Four key phenylalanine residues (F68, F142, F143, F179) were identified as vulnerable to radical attack.
- Single mutants showed only marginal improvement in radical stability.
- A quadruple mutant (F68A/F142A/F143A/F179A) retained 41% activity, a significant enhancement over the completely inactivated wild-type.
Conclusions:
- Engineering out multiple radical-vulnerable phenylalanine residues is an effective strategy to improve peroxidase stability.
- This approach offers a promising solution for overcoming limitations in industrial peroxidase applications.
Keywords:
horseradish peroxidasemass spectrometryradical couplingradical stabilitysite-directed mutagenesisMore Related Videos
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