BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes Christiane Liers & Caroline Bobeth & Marek Pecyna & René Ullrich & Martin Hofrichter Received: 17 June 2009 / Revised: 27 July 2009 / Accepted: 28 July 2009 # Springer-Verlag 2009 Abstract The jelly fungus Auricularia auricula-judae pro- duced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l -1 ). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l -1 . Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as “dye-decolorizing peroxidases”. Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylthiazoline-6- sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn 2+ ) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases. Keywords Dye-decolorizing peroxidases . Jelly fungi . White rot . Nonphenolic β-O-4 lignin model compound . Azo and anthraquinone dyes Introduction Most heme peroxidases belong to the superfamily of plant and microbial peroxidases. Based on the amino acid sequence consensus as well as on functional and structural properties, this superfamily is divided into three major classes: class I contains intracellular peroxidases of prokaryotic origin, class II includes secretory fungal peroxidases like the lignin- degrading enzymes manganese peroxidase (MnP), lignin peroxidase (LiP) or versatile peroxidase (VP); and class III harbors plant peroxidases like horseradish (HRP), soy bean (SBP), tobacco (TOP) or barley grain peroxidases. As a characteristic feature, heme peroxidases possess a ferriprotoporphyrin IX as prosthetic group at the active site and ten conserved α-helices. They are able to oxidize various organic and some inorganic compounds (Mn 2+ , I - , Br - ) using hydrogen peroxide as electron acceptor while reducing it to water (Welinder 1992; Morgenstern et al. 2008) though this classification applies to most of the known fungal peroxidases, meanwhile new families of fungal heme peroxidases have been discovered. They possess atypical protein sequences combined with unique catalytic properties, which complicates their classification into one of the three classes of the superfamily. For example, chloroperoxidase of the asco- mycete Caldariomyces fumago represents an “outgroup” of heme peroxidases and has an isolated position until recently; then other heme-thiolate peroxidases have been described for the agaric basidiomycetes Agrocybe aegerita C. Liers (*) : C. Bobeth : M. Pecyna : R. Ullrich : M. Hofrichter Unit of Environmental Biotechnology, International Graduate School of Zittau, Markt 23, 02763 Zittau, Germany e-mail: liers@ihi-zittau.de Appl Microbiol Biotechnol DOI 10.1007/s00253-009-2173-7