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)