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 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 recalcitrant AQ.

Dihydroxyacetone (DHA) is used as the main agent of self-tanning formulations. However, it is difficult to formulate DHA with high content because of its very high reactivity and instability, and it is known that coloring strength has been increased by using polyhydric alcohol in combination up to now. In this study, we investigated improving coloring strength in vitro and in vivo using water-soluble components including polyhydric alcohol. As a result, Methyl gluceth-10 (MG-10) was most effective in vitro． In contrast, MG-10 alone was the most ineffective in vivo． However, it was revealed that the coloring strength was improved by mixing with ethanol. We thought that ethanol distrubs the lamellar structure in the stratum corneum, shakes the intercellular lipid structure, and makes skin permeation easier, whereas the molecular weight was so large that it could not penetrate the skin alone. We focused on the polyethylene glycol (PEG) structure in MG-10, and changed the PEG chain length in vitro to confirm the coloring effect. Furthermore, as an additional test, the number of oxygen atoms in the solution was adjusted to PEG-40 and made constant, and the coloring effect was confirmed. As a result, in the test in which the number of moles of PEG was constant, it was found that the coloring effect was improved as the chain length of PEG became longer. On the other hand, in the test in which the number of oxygen atoms in the solution was constant, the same absorbance was obtained uniformly. From these results, it was thought that the oxygen in the PEG chain contributed to the coloring effect. Furthermore, MALDI／MS measurement and FT-IR measurement suggested that oxygen atoms in the PEG chain plays the role of mediator via its hydrogen bond with the amino acid and DHA.