BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues Henry Joseph Oduor Ogola & Naoya Hashimoto & Suguru Miyabe & Hiroyuki Ashida & Takahiro Ishikawa & Hitoshi Shibata & Yoshihiro Sawa Received: 5 March 2010 / Revised: 31 March 2010 / Accepted: 4 April 2010 / Published online: 27 April 2010 # Springer-Verlag 2010 Abstract Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacte- rium 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 H 2 O 2 . The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H 2 O 2 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 H 2 O 2 , respectively, in addition to maintaining high dye- decolorization activity toward AQ and azo dyes at 5 mM H 2 O 2 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 recalci- trant AQ. Keywords DyP-type peroxidase . Oxidative stability . Methionine residue . Site-directed mutagenesis . Bioremediation Introduction Peroxidases are heme-containing enzymes that use H 2 O 2 as the electron acceptor to catalyze numerous oxidative reactions and are potentially useful for practical applica- tions because of their broad spectrum of activities. In particular, their ability to degrade a wide range of substrates using the “clean” oxidant H 2 O 2 has made them potentially useful for diagnostic and bioindustrial applications (Colonna et al. 1999), as well as environmental bioreme- diation of recalcitrant and xenobiotic wastes (Ollikka et al. 1993; Robinson et al. 2001; Christian et al. 2003). Dye- decolorizing peroxidases (DyPs), a novel family of heme- containing peroxidases named after their capability to efficiently oxidize the high redox potential trichromatic anthraquinoic dyes (AQ), have been identified in various basidiomycetes ( Kim and Shoda 1999; Johjima et al. 2003; Faraco et al. 2006; Scheibner et al. 2008; Liers et al. 2010) and bacterial species (Sturm et al. 2006; Zubieta et al. 2007; van Bloois et al. 2009). DyPs have been the focus of significant interest due to their high specificity for AQ, low pH optima, dramatic differences in their active site topology, heme-binding motif, and structural divergence Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2603-6) contains supplementary material, which is available to authorized users. H. J. O. Ogola : N. Hashimoto : S. Miyabe : T. Ishikawa : H. Shibata : Y. Sawa (*) Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan e-mail: ysawa@life.shimane-u.ac.jp H. Ashida Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, Matsue, Shimane, Japan Appl Microbiol Biotechnol (2010) 87:1727–1736 DOI 10.1007/s00253-010-2603-6