Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace f...

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Bibliographic Details
Published in:	Applied microbiology and biotechnology Vol. 87; no. 5; pp. 1727 - 1736
Main Authors:	Ogola, Henry Joseph Oduor, Hashimoto, Naoya, Miyabe, Suguru, Ashida, Hiroyuki, Ishikawa, Takahiro, Shibata, Hitoshi, Sawa, Yoshihiro
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-08-2010 Springer-Verlag Springer Springer Nature B.V
Subjects:	Amino Acid Substitution - genetics Anabaena Anabaena - enzymology Azo dyes Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Binding sites Biological and medical sciences Biomedical and Life Sciences Bioremediation Biotechnologically Relevant Enzymes and Proteins Biotechnology Catalysis Cloning Cyanobacteria Dyes DyP-type peroxidase E coli Enzyme Stability Enzymes Fundamental and applied biological sciences. Psychology Hydrogen peroxide Hydrogen Peroxide - metabolism Inactivation Life Sciences Methionine - genetics Methionine residue Microbial Genetics and Genomics Microbiology Models, Molecular Mutagenesis Mutagenesis, Site-Directed Mutant Proteins - chemistry Mutant Proteins - genetics Mutant Proteins - metabolism Mutation Oxidation oxidative stability Peroxidase - chemistry Peroxidase - genetics Peroxidase - metabolism Protein Stability Protein Structure, Tertiary Proteins Residues site-directed mutagenesis Studies Substrate Specificity Wastewater pollution Water treatment DyP-type peroxidase Methionine residue Bioremediation Oxidative stability Site-directed mutagenesis Hydrogen peroxide Stability Enzyme Site directed mutagenesis Methionine Peroxidases Residue Cyanobacteria Anabaena Bacteria Peroxidase Oxidoreductases
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Summary:	Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H₂O₂. The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H₂O₂ stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H₂O₂, respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H₂O₂ and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.
Bibliography:	http://dx.doi.org/10.1007/s00253-010-2603-6 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0175-7598 1432-0614
DOI:	10.1007/s00253-010-2603-6