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Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack

Paper ID Volume ID Publish Year Pages File Format Full-Text
23064 43415 2014 8 PDF Available
Title
Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack
Abstract

•An adduct between F230 and a phenoxyl radical in inactivated CiP was identified.•F230A mutant showed the improved stability against radical inactivation.•F230A mutant exhibited the enhanced turnover of the phenol substrate.•F230A mutant was stable during the oxidation of m-cresol and 3-methoxyphenol.•The novel strategy was proposed to develop the radical-stable peroxidases.

Despite the potential use of peroxidases as industrial biocatalysts, their practical application is often impeded due to suicide inactivation by radicals generated in oxidative reactions. Using a peroxidase from Coprinus cinereus (CiP) as a model enzyme, we revealed a dominant factor for peroxidase inactivation during phenol oxidation, and we engineered radical-stable mutants by site-directed mutagenesis of an amino acid residue susceptible to modification by phenoxyl radical. Mass spectrometry analysis of inactivated CiP identified an adduct between F230 and a phenoxyl radical, and subsequently, the F230 residue was mutated to amino acids that resisted radical coupling. Of the F230 mutants, the F230A mutant showed the highest stability against radical inactivation, retaining 80% of its initial activity, while the wild-type protein was almost completely inactivated. The F230A mutant also exhibited a 16-fold higher turnover of the phenol substrate compared with the wild-type enzyme. Furthermore, the F230A mutant was stable during the oxidation of other phenolic compounds, including m-cresol and 3-methoxyphenol. No structural changes were observed by UV–vis and CD spectra of CiP after radical coupling, implying that the F230–phenol radical adduct inactivated CiP by blocking substrate access to the active site. Our novel strategy can be used to improve the stability of other peroxidases inactivated by radicals.

Keywords
Peroxidase inactivation; Radical coupling; Mass spectrometry; Protein engineering; Radical-stable peroxidase
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Development of the radical-stable Coprinus cinereus peroxidase (CiP) by blocking the radical attack
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 189, 10 November 2014, Pages 78–85
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us