Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus

AMB Express, Aug 2017

The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L−1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6–dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

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Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus

Lauber et al. AMB Expr Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus Christiane Lauber 0 Tatiana Schwarz 2 Quoc Khanh Nguyen 2 Patrick Lorenz 2 Guenter Lochnit 1 Holger Zorn 0 3 0 Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen , Heinrich-Buff-Ring 17, 35392 Giessen , Germany 1 Institute of Biochemistry, Justus Liebig University Giessen , Friedrichstrasse 24, 35392 Giessen , Germany 2 AB Enzymes GmbH , Feldbergstrasse 78, 64293 Darmstadt , Germany 3 Bioresources Project Group, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) , Winchesterstrasse 2, 35394 Giessen , Germany The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L−1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6-dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family. Pleurotus sapidus; Dyp-type peroxidases; White rot; β-carotene; Anthraquinone dyes; Lignin degradation Introduction Heme peroxidases have been classified into various superfamilies according to their functional and structural properties (Morgenstern et  al. 2008) . According to the classification of Welinder (1992) DyP-type peroxidases were assigned to Class II of the plant-peroxidase superfamily. This class includes the secretory fungal peroxidases and is characterized by a wide homogeneity; for example the manganese peroxidases (MnP), lignin peroxidases (LiP) and the versatile peroxidases (VP) all belong to this class (Lundell et al. 2010; Martíınez 2002) . All class II peroxidases are extracellular and contain heme as the prosthetic group (Poulos 2010; Welinder 1992) . The DyPtype peroxidases, however, show no homology to any other known peroxidase families. They possess a unique characteristic that differentiates them from other heme peroxidases and thus they consequently form their own superfamily (EC 1.11.1.19) among the heme-peroxidases. Recently Zámocký et  al. (2009, 2015) have suggested a new classification based on the overall fold, the structure of the active center and enzymatic activity. DyPtype peroxidases are now consequently allocated to the peroxidase-cyclooxygenase superfamily that is characterized by ferredoxin-like folding of the β-sheet structure and represents part of the very large α/β-barrel structure superfamily. The first indication of the existence of this peroxidase type was discovered by Kim et al. (1995 ). The first enzyme of this family (Bad DyP) was extracted from the fungus Bjerkandera adusta and was consequently purified and characterized (Kim and Shoda 1999a) . In the meantime DyP-type peroxidases have not only been discovered in Basidiomycota, but also in Ascomycetes and bacteria (Hofrichter et  al. 2010) . This also implies that these peroxidases have a common origin before the division of the domains (Sugano 2009) . The classical DyP from Bjerkandera adusta is the most completely characterized member of the family of DyP-type peroxidases. The term dye decolorizing peroxidase “DyP” presently describes a more polyphyletic group of enzymes (Ahmad et  al. 2011) . A subdivision of the peroxidases into three groups (P, I, V) has recently been suggested for the classification of DyP-type peroxidases (Yoshida and Sugano 2015) . Ten DyP-type peroxidases from fungi have been thus far characterized, and only those from B. adusta und A. auricula-judae have been characterized from a structural and mechanical perspective (Linde and Coscolñas 2014; Linde et al. 2015a; Strittmatter et al. 2013; Sugano 2009; Yoshida et  al. 2011, 2012) . DyP–type peroxidases possess a unique H2O2-bi (...truncated)


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Christiane Lauber, Tatiana Schwarz, Quoc Khanh Nguyen, Patrick Lorenz, Guenter Lochnit, Holger Zorn. Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus, AMB Express, 2017, pp. 164, Volume 7, Issue 1, DOI: 10.1186/s13568-017-0463-5