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

Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of... 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 −1 55,000 U L . 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 H O by rPsaDyP, however enzyme activity was increased by the 2 2 addition of H O . This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a 2 2 consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family. Keywords: Pleurotus sapidus, Dyp-type peroxidases, White rot, β-carotene, Anthraquinone dyes, Lignin degradation Introduction prosthetic group (Poulos 2010; Welinder 1992). The DyP- Heme peroxidases have been classified into various type peroxidases, however, show no homology to any superfamilies according to their functional and struc- other known peroxidase families. They possess a unique tural properties (Morgenstern et  al. 2008). According to characteristic that differentiates them from other heme the classification of Welinder ( 1992) DyP-type peroxi- peroxidases and thus they consequently form their own dases were assigned to Class II of the plant-peroxidase superfamily (EC 1.11.1.19) among the heme-peroxidases. superfamily. This class includes the secretory fungal per - Recently Zámocký et  al. (2009, 2015) have suggested a oxidases and is characterized by a wide homogeneity; for new classification based on the overall fold, the struc - example the manganese peroxidases (MnP), lignin perox- ture of the active center and enzymatic activity. DyP- idases (LiP) and the versatile peroxidases (VP) all belong type peroxidases are now consequently allocated to the to this class (Lundell et al. 2010; Martíınez 2002). All class peroxidase-cyclooxygenase superfamily that is character- II peroxidases are extracellular and contain heme as the ized 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 *Correspondence: holger.zorn@uni-giessen.de 1 peroxidase type was discovered by Kim et al. (1995). The Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Heinrich-Buff -Ring 17, 35392 Giessen, Germany first enzyme of this family (Bad DyP) was extracted from Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lauber et al. AMB Expr (2017) 7:164 Page 2 of 15 the fungus Bjerkandera adusta and was consequently As a result of their wide substrate spectrum DyP-type purified and characterized (Kim and Shoda 1999a). In peroxidases are interesting tools for biotechnological the meantime DyP-type peroxidases have not only been processes used in the decomposition of xenobiotic anth- discovered in Basidiomycota, but also in Ascomycetes raquinone derivatives in sewage and soil (Husain 2006; and bacteria (Hofrichter et  al. 2010). This also implies Sugano et al. 2007) and are already finding use in the food that these peroxidases have a common origin before the industry. For example, the DyP-type peroxidase MsP1 division of the domains (Sugano 2009). The classical DyP from M. scorodonius (Scheibner et al. 2008), marketed as from Bjerkandera adusta is the most completely charac- MaxiBright (DSM, Heerlen, the Netherlands), is used terized member of the family of DyP-type peroxidases. to whiten whey for cheese production. The enzyme thus The term dye decolorizing peroxidase “DyP” presently represents an alternative to chemical bleaches (Szweda describes a more polyphyletic group of enzymes (Ahmad et al. 2013). et  al. 2011). A subdivision of the peroxidases into three In the present report we publish a cDNA sequence that groups (P, I, V) has recently been suggested for the clas- codes for a DyP-type peroxidase from Pleurotus sapi- sification of DyP-type peroxidases (Yoshida and Sugano dus. Only two reports have been published on the DyPs 2015). from Pleurotus fungi (Faraco et  al. 2007; Fernández- Ten DyP-type peroxidases from fungi have been thus Fueyo et  al. 2015). The enzyme was efficiently expressed far characterized, and only those from B. adusta und A. heterologously in Trichoderma reesei, purified and bio - auricula-judae have been characterized from a structural chemically characterized. Homology studies allow the and mechanical perspective (Linde and Coscolñas 2014; identification of important catalytic amino acids and Linde et  al. 2015a; Strittmatter et  al. 2013; Sugano 2009; potential substrate oxidation sites. Yoshida et  al. 2011, 2012). DyP–type peroxidases pos- sess a unique H O -binding site that differs from those Materials and methods 2 2 of other peroxidases (Sugano et al. 1999). Whereas in the Chemicals enzymes of the superfamily of plant peroxidases the rear- Chemicals and reagents used were obtained from Carl rangement of the protons of H O is typically mediated Roth (Karlsruhe, Germany), Sigma (Neu-Ulm, Germany) 2 2 by the distal histidine this is accomplished by an aspar- or Merck (Darmstadt, Germany). Chemicals and materi- tate in DyP-type peroxidases (Sugano et al. 2007). als for electrophoresis were from Serva and Bio–Rad. DyP-type peroxidases are catalytically very versatile and stable (Pühse et al. 2009). They possess a high redox Strains and culture methods potential (1.1–1.2  V) so that numerous substrates can Pleurotus sapidus (DSM 8266) was obtained from the serve as electron donors (Liers et  al. 2013a). DyP-type German Collection of Microorganisms and Cell Cultures peroxidases are therefore able to oxidize a wide spectrum (DSMZ, Braunschweig, Germany). The Basidiomycete −1 of complex dyes, in particular xenobiotic anthraquinone was cultivated in standard culture medium (30 g L d– −1 −1 derivatives as well as typical peroxidase substrates such (+)–glucose·H O, 4.5  g  L l-asparagine·H O, 1.5  g  L 2 2 −1 −1 as ABTS (2,2′-azino-bis(3-ethylthiazoline-6-sulfonate)) KH PO , 0.5  g  L MgSO ·H O, 3.0  g  L yeast extract, 2 4 4 2 −1 −1 and phenols (Sugano 2009). Certain Dyp-type peroxi- 1 mL L trace element solution: 5 mg L CuSO ·5H O, 4 2 −1 −1 dases cleave β-carotene and other carotenoids, non-phe- 80  mg  L FeCl ·6H O, 90  mg  L ZnSO ·7H O, 3 2 4 2 −1 −1 nolic bonds such as veratryl alcohol and the β-O-4 lignin 30 mg L MnSO ·H O, and 0.4 g L EDTA; pH 6.0) for 4 2 model dimer adlerol (Liers et al. 2010). 10 days (24 °C, 150 rpm, 25 mm shaking diameter). Pleurotus sapidus is a Basidiomycete of the family Pleu- Escherichia coli (TOP10) was obtained from Invitrogen rotaceae (oyster mushrooms) belonging to the white-rot (Karlsruhe, Germany) and was used for vector propa- fungi. It grows on wood (pillar fungus). White-rot fungi gation. TSS competent cells were transformed by heat are the most efficient lignin destruents and are able to shock treatment (2  min, 42  °C) according to the stand- fully decompose complex polymers (Rajarathnam et  al. ard protocols. Recombinant cells were cultivated in ster- −1 −1 1998). In addition, P. sapidus is able to decompose natu- ile LB medium (10 g L tryptone, 5 g L yeast extract, −1 −1 ral pigments such as β-carotene (Schüttmann et al. 2014). 10 g L NaCl) with 150 mg L ampicillin used as selec- Numerous enzymes that are involved in lignocellulose tion marker (37 °C, 225 rpm). decomposition have been identified in the secretome of P. sapidus. For example, the fungus secretes cellulases, cDNA‑synthesis hemicellulases, peptidases, esterases, laccases and in par- To isolate the RNA the mycelium of a P. sapidus sub- ticular peroxidases into the culture medium (Zorn et  al. mersion culture was harvested on culture day 5 and 100 2005). mg was disintegrated by grinding in liquid nitrogen. Lauber et al. AMB Expr (2017) 7:164 Page 3 of 15 TM 2, BioTek Instruments GmbH, Bad Friedrichshall, Isolation of the total RNA was performed with RNeasy Germany) or alternatively in a UV/Vis spectrophotom- Plant Mini Kits (Qiagen, Hilden, Germany) according to eter (SPECORD 50, Analytik Jena AG, Jena, Germany). the manufacturer’s instructions. The quality of the RNA Reactions were initiated by addition of the enzyme. was verified by agarose electrophoresis and ethidium Enzyme activity was measured over a period of 10  min bromide staining. A cDNA library of P. sapidus was pro- at 25  °C at the appropriate wavelength for the substrate. duced using the isolated RNA as template. cDNA syn- One unit (1 U) is defined as the amount of enzyme that thesis was performed with help of the SMART PCR converts 1  µmol substrate per minute. Various H O cDNA Synthesis Kits (Clontech Laboratories Inc., Saint- 2 2 concentrations (0–1250  µM, enzyme concentrations Germain-en-Laye, France). SuperScript   II Reverse (0.27–54 nM) and substrate concentrations were used to Transcriptase (Invitrogen, Darmstadt, Germany) was determine the enzyme activity. used for first-strand synthesis. Amplification of a cod - The activity of rPsaDyP vs ABTS was determined in ing DyP-type peroxidase sequence of P. sapidus from 100 mM sodium acetate buffer at pH 3.8 and a final H O the cDNA library was performed by PCR. The prim - 2 2 concentration of 125 µM. Production of the ABTS cation ers DTP_for58 (5′-ATGCGCTGGTGGACTACC-3′) radical was studied according to Eggert et  al. (1996) at and DTP_rev54 (5′-TTAAGCAGCGATTTTGTGC-3′) −1 −1 420 nm (ε 36,000 L mol  cm ). were derived from a homologous Dyp-type peroxidase Oxidation of DMP and guaiacol was followed in sequence from the genome (DOE-JGI) of Pleurotus 50  mM sodium acetate buffer at pH 4.5 at a final H O ostreatus. Amplification of the specific cDNA occurred 2 2 concentration of 62.5  µM as based on the forma- in an Alpha SC PCR Thermocycler (Analytik Jena, Jena, tion of the dimeric quinone derivate at 469  nm (ε Germany). The following PCR protocol was followed: −1 −1 27,500  L  mol   cm referred to the substrate) accord- 50  ng template, 5×  PCR-Puffer including dNTP’s (Qia - ing to Saparrat et  al. (2002) or tetraguaiacol at 470  nm gen), forward and reverse primer 50  pmol, 1.25  U Hot- −1 −1 (ε 26,600 L mol  cm ) according to Koduri and Tien Star HiFidelity DNA-Polymerase (Qiagen) ddH O ad 2 470 (1995). 50  µL, 95  °C 5  min–95  °C 1  min, 51  °C 1  min, 72  °C Oxidation of Reactive Blue  5 dye (Rblue 5) was per- 2 min, 40 cycles–72 °C 5 min. PCR products were sepa- formed in 100  mM sodium acetate buffer at pH 4.0 rated electrophoretically (1.2% (w/v) agarose gel), subse- and a final H O concentration of 31.2  µM. Degrada- quently isolated from the gel using NucleoSpin Extract 2 2 tion of the substrate was determined at 600  nm (ε II–Kits (Machery-Nagel, Düren, Germany) and finally −1 −1 8000 L mol  cm ) according to Sugano et al. (2006). ligated (Topo TA-Cloning Kit, Invitrogen) in the vec- Oxidation of the azo dye Reactive Black 5 (RBlack 5) tor pCR2.1–TOPO (Invitrogen). The plasmid DNA was was determined in 50 mM sodium acetate buffer at pH 4.0 replicated in E.  coli TOP10 cells (Invitrogen), isolated −1 −1 with 62.5 µM H O at 598 nm (ε 37,200 L mol  cm ) from the cells and purified (NucleoSpin Plasmid DNA 2 2 598 according to Sugano et al. (2006). Purification, Machery-Nagel). Sequencing of the cloned A β-carotene stock solution was prepared as described cDNA was performed by MWG Eurofins (Ebersberg, by Pühse et  al. (2009). Measurement of the substrate Germany). degradation was performed at 450  nm (ε 95,000 −1 −1 L  mol   cm ) according to Ben Aziz et  al. (1971) in Enzyme production 50 mM sodium acetate buffer at pH 3.5 with a final H O For recombinant production of the peroxidase the codon 2 2 concentration of 125 µM. To assess the purified enzyme’s usage of the gene was adapted to the host organism oxygenase activity in the absence of H O , 100  mL Trichoderma reesei (a derivative of RUT C30; Peterson 2 2 sodium acetate buffer (100 mM, pH 3.8) was purged with and Nevalainen 2012) and an expression cassette was oxygen through a glass frit (pore size 3, pore diameter constructed from the adapted sequence. The expression 16–40  μm) for 30  min. For control experiments in the of peroxidase took place under the control of the cbhl absence of H O and oxygen, 100  mL of the buffer was promotor and terminator. Efficient production of the 2 2 sonicated for 60  min, and residual oxygen was removed peroxidase was achieved by optimization of the fermen- by purging with nitrogen. The assays were performed tation process under fed-batch conditions (pH5.5, 28 °C, with enzyme concentrations of 270, 360, and 540  nM, 160 h) in Monosaccharide medium (7% monosaccharide, and the concentration of the substrate was 26 µM. 4% agricultural waste stream derived N-source, 0.7% As further examples of natural dyes annatto (aque- (NH ) SO , 0.3% KH PO ). 4 2 4 2 4 ous alkaline extract), the principle component of which is norbixin, and bixin were used as substrates. Prepara- Determination of enzyme activity tion of the bixin stock solution was analogous to that of Enzyme activity was determined photometrically using a β-carotene (using 15  mg bixin). Conversions took place temperature controlled multi-mode plate reader (Synergy Lauber et al. AMB Expr (2017) 7:164 Page 4 of 15 in 50  mM sodium acetate buffer at pH 6.0 or 3.5 with a of the proteins took place in a 6% stacking and a 12% sep- final H O concentration of 125  µM. Degradation of the aration gel. Protein bands were visualized using colloidal 2 2 −1 −1 dye was observed at 452 nm (ε 108,400 L mol  cm , Coomassie staining (Kang et al. 2002). Size determination −1 −1 ™ Scotter et al. 1998) or 465 nm (ε 136,100 L mol  cm , was performed with the help of PageRuler Unstained Hülsdau 2007). Proteinladder (Fermentas, St. Leon–Rot, Germany). For Samples of heat inactivated (95  °C, 10  min) enzyme N-deglycosylation samples were incubated with PNGase served as negative controls. F (NEB, Ipswich, USA) and treated following the manu- facturer’s instructions. Purification of the DyP‑type peroxidase by fast protein The size of the native proteins was determined by gel liquid chromatography (FPLC) filtration chromatography (GFC) and Blue Native PAGE. Purification was carried out in a cold room at 4 °C. Chro - For GFC the protein was loaded onto a Superdex 200 matographic separation of the proteins was performed 10/300 GL column (GE Healthcare) and eluted with using a BioLogic DuoFlow (Biorad) FPLC–System with a 50  mM sodium acetate buffer pH 4.0 with 250  mM fraction collector (BioLogic BioFrac fraction collector, NaCl and 0.005% Triton X-100 at a flow rate of 0.5  mL −1 Biorad). Proteins were detected at 280 nm. The DyP-type min . For Blue Native PAGE precast gels from Serva peroxidase was purified in a two-stage process by hydro - (SERVAGel N Native Starter Kit, Serva, Heidelberg, phobic interaction chromatography and ion exchange Germany) and the PerfectBlue Double Gel System Twin chromatography. The culture supernatant was concen - S of Peqlab were used. For isoelectric focusing (IEF) the trated (Macrosep , 10  kDa cutoff, Pall, Dreieich, Ger - same electrophoresis system was used (precast vertical many) and transferred to 50  mM sodium acetate buffer gels and IEF marker 3–10, Liquid Mix, Serva). Visuali- at pH 4.0 with 1 M (N H ) SO . In the first stage the con - zation of the protein bands was performed as described 4 2 4 centrated culture supernatant was purified on a column above. In addition, specific staining of heme- and metal with phenyl-Sepharose-matrix (HiPrep Phenyl FF (high enzymes was performed with 3,3′,5,5′-tetramethylb- −1 sub) 16/10, GE Healthcare Bio–Sciences AB, Uppsala, enzidine (TMB) (1.5  mg  mL in methanol) modi- Sweden). The Starting buffer used was 50  mM sodium fied according to Thomas et  al. (1976) and Henne et  al. acetate buffer pH 4.0 with 1  M (NH ) SO and proteins (2001). Native electrophoresis was followed by activ- 4 2 4 were eluted over a gradient of 0 to 58% (40  mL) and ity staining with ABTS as substrate. For this the gel was from 58 to 100% (30  mL) sodium acetate buffer pH 4.0. equilibrated for 5  min in 100  mL freshly prepared stain −1 Flow rate was 3  mL  min and fractions with a volume solution (5 mM ABTS in 50 mM sodium acetate pH 4.0) of 2 mL were collected. Protein containing fractions were on an orbital shaker. Subsequently, 150 μL 3% H O (final 2 2 tested and active fractions pooled for the second purifi - concentration 130  μM) was added and shaking contin- cation step. They were then concentrated, transferred to ued until a distinct staining was obtained. Gels were then 50  mM sodium acetate buffer pH 4.0 and loaded onto a rinsed with pure water and documented. column with SP Sepharose Fast Flow Matrix (XK16/20, 25 mL column volume, GE Healthcare Bio–Sciences AB). Influence of pH value Elution was performed on a gradient of 0–10% (30  mL) The optimum pH of the purified rPsaDyP was deter - and from 10 to 100% (30  mL) 50  mM sodium acetate mined in McIlvaine–Puffer (pH 2.2–7.5, McIlvaine, −1 buffer pH 4.0 with 1 M NaCl at a flow rate of 3 mL min . 1921), 50  mM sodium acetate buffer (pH 3.0–6.0) and Active fractions of the second purification stage were 100 mM sodium tartrate buffer (pH 2.0–5.5) with ABTS pooled, concentrated, aliquoted, shock frozen in liquid as substrate. The pH stability was determined by dilut - nitrogen and stored at −80 °C. ing the DyP-type peroxidase in sodium acetate buffer at a pH range of 3.0–6.0 (0.5 steps). To avoid altering the Enzyme characterization pH value too drastically for the measurement the sam- Protein concentration was determined according to ples were again diluted 1:10 with 100 mM sodium acetate Bradford (1976) using the dye solution Roti Nanoquant buffer pH 3.8 (measurement buffer). Rest activity was (Roth, Karlsruhe, Germany) with bovine serum albumin measured after 0.5, 1, 2 and 24  h storage at 4  °C using or lyophilized DyP as standard. ABTS as substrate and values were compared to the ini- UV/Vis spectra of the purified rPsaDyP were recorded tial activity. at 250 nm to 800 nm in a NanoPhotometer (Pearl, Implen GmbH, Munich, Deutschland). Eec ff t of temperature Molecular mass determination of the DyP-type peroxi- To determine the optimum pH for the purified rPsaDyP dase was performed using SDS–PAGE (Mini–Protean the enzyme was initially incubated for 4  min in 50  mM TetraSystem, BioRad) according to Laemmli. Separation sodium acetate buffer at pH 3.8 at various temperatures Lauber et al. AMB Expr (2017) 7:164 Page 5 of 15 (15–75  °C). The rate of turnover was then measured at Structure and sequence based analysis the corresponding temperature in a photometer with A structural homology model of the DyP-type peroxidase temperature regulated cuvette. from P. sapidus was calculated based on the x-ray crystal structure of DyP-type peroxidase from A. auricula-judae Determination of kinetic parameters (PDB-ID 4AU9B). The structures exhibit a homology of The apparent value of the Michaelis–Menten constant 44%. (K ) and catalytic constant (k ) of the purified rPsaDyP The calculated structural model possesses a helical m cat were determined for ABTS, DMP, guaiacol and RBlue 5. basic structure with two domains (proximal N-termi- Substrate turnover took place at 30 °C in 6 replicate prep- nal, distal C-terminal domains), that enclose the central arations. The initial rate was determined with constant cofactor heme. A prominent motif made up of anti-par- enzyme concentration, varying substrate concentrations allel β-sheet structures that, together with the α-helices, and constant cosubstrate concentration. Measurements exhibit a ferredoxin-like folding is present on the distal were taken in parallel with varying H O concentrations side of the heme. With the help of this structure homol- 2 2 in order to determine the H O concentration that no ogy model it was possible to identify important func- 2 2 longer had a limiting effect on the activity. The follow - tional amino acid residues (Fig.  2). The access channel ing substrate concentrations were chosen for determi- to the heme of the catalytic channel forms a predomi- nation of the kinetic parameters: ABTS 15–1500  µM, nantly hydrophobic channel through which the sub- DMP 250–15,000 µM, guaiacol 500–15,000 µM, RBlue 5 strate reaches the H O binding pocket (Yoshida et  al. 2 2 11–300 µM. The initial rate (ν ) was plotted linear to the 2011). Three of the remaining residues of the H O bind- 0 2 2 substrate concentration in a graph (Cornish-Bowden ing pocket are also conserved in rPsaDyP, whereby in diagram). Using the software Origin (OriginPro 8.6G) a the DyP-type peroxidases from P. sapidus and P. ostrea- saturation hyperbola was adapted through a non-linear tus leucine is exchanged for valine (rPsaDyPV363). regression in order to obtain values for K and ν . The The catalytic residues of D174—part of the conserved m max catalytic constant k was determined using the following GxxDG motif—and R338 on the proximal side H317 as cat equation, whereby E represents the enzyme concentra- fifth ligand of heme were identified. These residues are tion applied: involved in the activation of the enzyme (formation of compound I) by heterolytic cleavage of H O . Based on 2 2 max k = the alignment (Fig.  2) it was shown that E391, which cat [E] forms a hydrogen bond with the fifth ligand of heme Results (H317), thus stabilizing compound I (Sugano et al. 2007), Identification of the coding sequence of DyP‑type is exchanged for an aspartate (rPsaDyP D401) in other peroxidase Dyp-type peroxidases. The arginine 267 and 324 hydro - The 1551 bp full-length cDNA of a DyP-type peroxidase gen bonds with the propionate residues of the heme gene of Pleurotus sapidus (strain 8266) was amplified. form additional ligands. The conserved amino acid resi - This gene codes for a protein of 516 AA (Fig.  1, Acces- dues Y343 and W383 that were exposed to the solvent sion Number LN830264), a theoretical molecular weight are found on the surface. These serve as oxidation sites of 57.1  kDa and a calculated isoelectric point (pl) of for large substrates and may be elements of a LRET (long 5.73 (ExPASy ProtParam). The ORF exhibits 4 potential range electron transfer) (Liers et  al. 2013a; Strittmatter N-glycosylation sites and a potential O-glycosylation site et al. 2013). (NetNGly 1.0, NetOGly3.1). With the identification of putative conserved domains Heterologous expression and purification the enzyme is assigned to the dye decolorizing peroxi- For purification and characterization peroxidase positive dase superfamily (EMBL-EBI, InterProScan). In addition, transformants were cultivated in large scale (XL) under the structural motif GxxDG that is present in all mem- conditions that yield active protein in the culture super- −1 bers of the DyP-type peroxidase family was found in the natant. After 160 h cultivation an activity of 55,000 U L sequence. The translated amino acid sequence shows a in relation to the substrate ABTS was achieved and the high degree of identity (95%) to a DyP-type peroxidase supernatant containing the peroxidase was harvested. from Pleurotus ostreatus (ID EMBL CAK55151.1). Fur- After concentration of the culture supernatant (crude thermore, the amino acid sequence exhibits an identity enzyme) by a factor of 5 the Dyp-type peroxidase was of 42–43% with DyP-type peroxidases from B. adusta purified in a two-stage sequential procedure using FPLC (Accession Number: BAA77283.1), M. scorodonius with hydrophobic interaction chromatography and ion (Accession Number: B0BK71.1) and A. auricula-judae exchange chromatography. The DyP-type peroxidase (Accession Number: AFJ79723). was purified to apparent homogeneity. After the second Lauber et al. AMB Expr (2017) 7:164 Page 6 of 15 Fig. 1 Nucleotide- and derived amino acid sequence of a DyP-type peroxidase from Pleurotus sapidus (LN830264). Predicted N-glycosylation sites are highlighted; a hypothetical cleavage site after 62 amino acids is marked with an arrow Lauber et al. AMB Expr (2017) 7:164 Page 7 of 15 Fig. 2 Alignment of various DyP-type peroxidase sequences from P. sapidus (PsaDyP), P. ostreatus (PosDyP, NCBI: CAK55151), B. adusta (BadDyP, NCBI: BAA77283), A. auricula-judae (AauDyP, NCBI: AFJ79723) and M. scorodonius (MsP1, NCBI: BOBK71). Conserved amino acids (*); the character- istic GxxDG motif is enclosed in a frame; remnants of the H O binding site (circle); E391 (blue); conserved residues of the heme binding site (gray 2 2 background); potential heme binding residues (triangle); exposed amino acids of a potential LRET (long range electron transfer, green background); remnants involved in a potential LRET (green); N-terminal conserved amino acid sequences (white); conserved histidine residues (H164 and H166, blue background); signal sequences (gray); N-terminal amino acids are underlined Lauber et al. AMB Expr (2017) 7:164 Page 8 of 15 purification step the active fractions eluted in a single, oligosaccharides from the N-glycosylation sites. After discreet peak (Fig.  3). Following the two-stage puri- treatment with the glycosidase rPsaDyP showed a lower fication an electrophoretic homogeneity of DyP was apparent molecular weight in SDS-PAGE than the −1 achieved with a yield of 2.9 ± 0.1 g L and an increase in untreated samples (52.8–57.6  kDa). This indicates that Reinheitszahl from 0.2 to 1.1. rPsaDyP is glycosylated by the host organism. From the difference in molecular weights a degree of glycosylation Characterization of rPsaDyP of ~9% was calculated. The purified enzyme showed a typical heme-enzyme red coloring and a maximum absorbance λ  = 409 nm and N‑terminus max two further maxima at 510 and 640 nm (Fig. 4). A molec- The detected apparent molecular weight of the deglyco - ular weight of 57.6  kDa was determined by SDS-PAGE. sylated rPsaDyP is lower than the theoretical weight that Isoelectric focusing (IEF) showed a pl of 6.7 (Fig. 4). was calculated from the primary sequence. This suggests that the protein is processed in the host organism. There - Determination of the native conformation fore, the N-terminus of the recombinant enzyme was The native conformation of the DyP was established by determined by Edman degradation. The first 12 residues Blue Native PAGE and gel filtration chromatography. of the N-terminal amino acid sequence of the purified By electrophoresis a molecular weight of 115  kDa was enzyme (AT(N)GTFLPLEEI) were identified. According ascertained for the native, active enzyme and the reten- to this the enzyme has a signal peptide 62 amino acids tion time of the peaks in gel filtration chromatography long and the mature protein a total length of 454 amino indicated an apparent molecular weight of 122 kDa. The acids. molecular weights indicated by these two methods were comparable and approximately double the value of the Catalytic properties of rPsaDyP apparent molecular weight of rPsaDyP under denaturing pH‑ and temperature optimum conditions (57.6  kDa). This suggests that the quaternary Before the kinetic constants were determined the optimal structure of the native enzyme is a dimer. pH for oxidation of the substrate investigated was identi- Four potential N-glycosylation sites were predicted fied (Fig.  5). The pH-optimum for the oxidation of ABTS in the amino acid sequence. The protein was therefore and Rblue 5 was found to be between 3.5 and 4.0, for the treated with PNGase F in order to remove any possible reaction with phenolic substrates the pH-optimum was Fig. 3 Cation-exchange chromatography as second purification step using an SP Sepharose FF column; ABTS oxidizing enzyme activity was detected in the area with a gray background; UV absorption in AU at 28 nm (blue line), detected activity (dashed line), buffer concentration (50 mM sodium acetate buffer pH 4.0 with 1 M NaCl) in % (light gray line) Lauber et al. AMB Expr (2017) 7:164 Page 9 of 15 Fig. 4 a UV–Vis absorption spectrum of the purified rPsaDyP showing the Soret band at 409 nm and two additional maxima in the region of 510 and 640 nm; b isoelectric focusing of purified rPsaDyP, stained with colloidal Coomassie (1) and specific staining for heme/metal enzymes with TMB (2); M IEF Marker 3–10 Fig. 5 a Optimum pH value for oxidation of ABTS, DMP and guaiacol by rPsaDyP in 50 mM sodium acetate buffer or Rblue 5 in 100 mM sodium tartrate buffer. b Eec ff t of temperature on the activity of rPsaDyP with ABTS as substrate higher (pH 4.5). In addition, the enzyme activity for the stability residual activity was detected after a 5 min incu- substrates was determined under varying H O concentra- bation of the enzyme. At temperatures over 70  °C the 2 2 tions (0–1250 µM). With ABTS as substrate the peroxidase enzyme completely lost activity. A 5–min T –Wert of activity fell significantly when the H O concentration rose 53  °C was determined from the residual activity of the 2 2 above 0.125 mM, indicating that the enzyme is inhibited by enzyme. Under assay conditions the enzyme showed a H O . Maximum reaction rates depending upon substrate 75% residual activity after 2  h and after 24  h 40% of the 2 2 tested reached values between 31.2 and 125 µM (31.2 µM: initial activity was still present. RBlue 5, 62.5  µM: DMP, guaiacol, RBlack 5; ABTS, Ann- atto, Bixin, β-carotene: 125 µM). Catalytic properties The enzyme showed maximum activity over a tem - The apparent kinetic constant for rPsaDyP (expressed perature range of 15–30  °C. To determine the thermal in T. reesei as active protein) was determined for ABTS, Lauber et al. AMB Expr (2017) 7:164 Page 10 of 15 RBlue 5, DMP and guaiacol as shown in Table 1. The per - Table 2 Degradation of  β-carotene by  the purified recom- binant DyP-type peroxidase from Pleurotus sapidus (in the oxidase efficiently oxidized the low redox potential dye absence of  H O ) under standard assay conditions, in oxy- ABTS. In addition, rPsaDyP catalyzed the degradation 2 2 gen saturated buffer, and under anoxic conditions of the dye Rblue5, a characteristic DyP substrate. The −1 enzyme binds this substrate at high affinity (K  = 24 μM) Enzyme concen‑ v (µM min ) tration (nM) and converts it efficiently. The substrate spectrum of Degassed buffer Standard assay Oxygen rPsaDyP also includes the substituted phenols DMP and conditions saturated buffer guaiacol. Both of these substrates show a high K (713 and 1227  µM, respectively) and a relatively low catalytic 270 0.045 0.385 0.878 efficiency compared to the other substrates. 360 0.056 0.495 1.069 DyP is also able to catalyze the degradation of the nat- 540 0.077 0.532 1.217 −1 ural pigment β-carotene ene (90  U  L ). A study of the kinetics of the conversion of this substrate was difficult, however, since only low substrate concentrations (maxi- mal 26  µM) could be used in the assay and thus satura- was cloned and sequenced. By comparing the converted tion of the enzyme could not be achieved. Notably, the amino acid sequence with other DyP-type peroxidases, DyP-type peroxidase can convert this substrate without and with the structural homology model of the structure the addition of H O in the same manner as MsP1 and of AauDyP as a basis, shared motifs and catalytic residues 2 2 MsP2 (Scheibner et  al. 2008; Zorn et  al. 2003b). At the could be identified. The model demonstrates the charac - same time, enrichment of the buffer with O increased teristic β-barrel structure and environment of the heme the conversion rate by a factor of 2.3 compared to stand- pocket. This includes the characteristic GxxDG motif ard conditions without addition of H O . The enzymatic with the conserved aspartate that, together with the con- 2 2 conversion was virtually halted when degassed buffer was served arginine residue is situated on the distal side of the used (Table  2). This indicates that the enzyme also pos - heme, as well as the proximal histidine. The alignment sesses oxidase or oxygenase activity. shows that the amino acids involved in the heme binding Furthermore, the enzyme can also convert additional as well as those involved in catalysis are highly conserved. natural pigments such as bixin and annatto (90 and DyP-type peroxidases are also capable of oxidizing −1 114  U  L ), as well as high redox potential dyes such as large substrates that are not able to reach the immediate −1 RBlack 5 (231 U L ). proximity of the heme in the active center. For this reason it is a matter of discussion as to whether these enzymes Discussion exclusively possess solvent-exposed substrate binding The first published sequence of a DyP-type peroxidase sites (Liers et al. 2013a). Strittmatter et al. (2013) identi- originated from the Basidiomycetes B. adusta (Kim and fied potential LRET transfer pathways in the DyP-type Shoda 1999b). In the meantime further DyP-type per- peroxidase from A. auricula-judae. A number of exposed oxidases from white-rot fungi have been cloned and residues on the protein surface (Trp or Tyr) serve as sequenced, including P. ostreatus, M. scorodonius und A. oxidation site for large substrates. Linde et  al. (2015a, auricula–judae (Faraco et al. 2007; Scheibner et al. 2008; b) recently showed that LRET from AauDyP essentially Liers et al. 2010). X-ray structural analysis has only been begins at W377 that is also conserved in rPsaDyP. performed on two DyP-type peroxidases from fungi. The histidine 164 or 166 (BadDyP) are conserved in The first DyP-peroxidase structure examined was from many DyP sequences. It was therefore long a matter of B. adusta (BadDyP; PDB-Code 2D3Q). In the meantime discussion whether these residues function as proximal the structure of a DyP from A. auricula-judae (AauDyP; histidine or as heme ligand. Sugano et al. (2004) showed PDB-Code 4AU9) has also been elucidated. In the pre- that H166 is not essential for the peroxidase activ- sent study the first DyP-type peroxidase from P. sapidus ity. On the other hand, the authors showed that mutant Table 1 Apparent kinetic constants of the recombinant DyP-type peroxidase from Pleurotus sapidus −1 −1 −1 −1 −1 Substrate pH Enzyme concentration (nM) K (µM) k (s ) k K (s  M ) v (µM s ) m cat cat m max ABTS 3.8 0.27 99 375 3.8 × 10 0.10 DMP 4.5 1.8 1227 60 4.9 × 10 0.11 Guaiacol 4.5 4.5 713 74 1.0 × 10 0.35 RBlue 5 4.0 5.4 24 18 7.5 × 10 0.10 Lauber et al. AMB Expr (2017) 7:164 Page 11 of 15 H164A completely lost the activity. This result indicates of the DyP-type peroxidase MsP2 (Scheibner et al. 2008). that H164 is not directly involved in heme binding, but The carbohydrate content of DyP-type peroxidases lies rather suggests only a decrease in protein stability and typically between 9 and 31% (Hofrichter et al. 2010). a loss of the heme-binding affinity (Faraco et  al. 2007). After deglycosylation the apparent molecular weight H164 is not conserved in rPsaDyP or in PosDyP, but is determined in SDS-PAGE was lower for rPsaDyP het- replaced by a lysine (K167). This exchange is also found erologously expressed in T. reesei than was calculated in various other representatives of the DyP-type per- from the primary sequence. This implies that the enzyme, oxidase family, for instance in the proteins of A. oryzae just as with BadDyP in A. oryzae (Sugano et  al. 2000), (Q2UPE9, Q2U1I3), Neurospora crassa (Q7S3A4) and is further processed in the host organism, T. reesei. The various other members of the DyP-family (Faraco et  al. N-terminus of the recombinant enzyme was sequenced 2007). This suggests that H164 is not directly involved in by Edman degradation. Processing of rPsaDyP is between heme binding and another residue coordinates the heme amino acids 62 and 63. Sugano et al. (2000) showed that in DyP-type peroxidases. Nonetheless, according to Sug- the recombinant DyP-type peroxidase from B. adusta ano (2009) H164 plays an important role in the folding of produced in A. oryzae has the same N-terminus as the DyP-type peroxidases and binding of heme, even if it is wild-type enzyme. This suggests that the recombinant not conserved in all members of this protein family. Here and native PsaDyP have the same N-terminus. Studies of the situation is different than described by the authors. other DyP-type peroxidases show that processing occurs Histidine does not appear to be crucial for folding, but between position 56/57 (BadDyP), 55/56 (MsP1), 57/58 rather, a basic amino acid in this position. Johjima et  al. (MsP2) and 61/62 (AauDyP) (Liers et  al. 2010; Scheib- (2003) identified 10 potential ligands (His, Tyr und Cys) ner et  al. 2008; Sugano et  al. 2000). In rPsaDyP a clus- for heme. For AauDyp Strittmatter et  al. (2013) showed ter of conserved amino acids is present in the region of that on the proximal side arginine 255 and 311 form the N-terminus, however a typical cleavage site was not hydrogen bonds to the propionate residues of heme and found (Liers et al. 2010). are involved in the coordination of heme. The homolo - In contrast to the classical DyP rPsaDyP occurs as gous residues from rPsaDyP were identified (at position a dimer, whereby it must be noted that various other 267 and 324) and in the model are at a distance of 3.2 and DyP-type peroxidases, especially from prokaryotes, 3.5 Å, respectively, to the propionates. form numerous higher quaternary structures ranging from monomers to hexamers. The reason for this oli - Heterologous expression gomerization has been subject of discussion, but remains The DyP-type peroxidase from Pleurotus sapidus was unknown. Sugano (2009) showed that the classical DyP, successfully expressed heterologously in the ascomy- compared to DyP-type peroxidases that form oligomers, cete T. reesei and the active enzyme was secreted into exhibit insertions in the primary sequence that are miss- −1 the culture supernatant. An activity of 55,000  U  L ing in the former. Nonetheless, the primary sequences was determined for the recombinant DyP-type peroxi- of MsP1 and MsP2 exhibit a high degree of homology to dase in the supernatant with the substrate ABTS. Het- BadDyP and like rPsaDyP and have these insertions, and erologous expression of the DyP from B. adusta in A. yet they occur natively as dimers (Scheibner et al. 2008). oryzae imparted a 42 fold increase in activity with the 2 −1 substrate RBlue 5 (8 × 10  U L ) over that in the culture Biochemical characterization medium (Sugano et al. 2000). Heterologous expression of DyP-type peroxidases are typically secreted glycoproteins the PsaDyP from T. reesei led to an order of magnitude with molecular weights of 40–67  kDa (in monomeric 3 −1 higher activity (5  ×  10  U L ) with RBlue 5. PsaDyP form) and isoelectric points in the acid range (3.5–4.3, could be efficiently expressed in T. reesei and unusu - Hofrichter et  al. 2010). A monomer of rPsaDyP has a ally high activities could be achieved. The recombinant molecular weight of 57.4 kDa and therefore is of average enzyme showed the characteristic absorption maximum size for an enzyme of the family of DyP-type peroxidases. at 409 nm (Soret–Band) and two further maxima (α and In contrast to other enzymes of this family rPsaDyP has β) in the region of 640 nm that are attributed to the por- an apparent Pl within the neutral range. Lignolytic per- phyrin structure of heme (Glenn and Gold 1985; Renga- oxidases from fungi (LiP, VP, DyP), like plant peroxi- nathan and Gold 1986). dases demonstrate maximum activity in an acidic milieu (pH 1.5–5.0, Camarero et  al. 1999; Gazarian et  al. 1996; Native conformation Liers et al. 2010; McEldoon et al. 1995). Depending upon A glycosylation degree of 9% was determined for the the substrate, the pH profile of rPsaDyP shows an activ - DyP-type peroxidase heterologously expressed in T. ree- ity maximum between pH 3.5 and 4. 5 (with the excep- sei, a carbohydrate content that is comparable with that tion of Annatto at pH 6.0). Thus, the profile is shifted to Lauber et al. AMB Expr (2017) 7:164 Page 12 of 15 somewhat higher pH values compared to other DyP-type for horseradish peroxidase. Compound III was reduced peroxidases. Maximum activity of rPsaDyP was shown to Compound I by various electron donors. A similar between 15 and 30  °C. This is comparable with that of substrate dependent mechanism may explain the diver- BadDyP (Kim and Shoda 1999a). Above 35 °C activity of gent inhibition of the enzyme by hydrogen peroxide in rPsaDyP decreased continuously and  ~65% remained at rPsaDyP. 50 °C. rPsaDyP is active in a wide range of temperatures and pH values. It remains active for a number of hours in Catalytic properties sodium acetate buffer between pH 3 and pH 6 and main - From the functional standpoint the heterologously tains up to 60% of its initial activity after 24  h (data not expressed peroxidase of P. sapidus can be assigned to shown). There is a tendency for the activity of the enzyme the class of DyP-type peroxidases as based on its sub- to remain stable at higher pH values. After 24  h under strate spectrum and the efficient oxidation of RBlue reaction conditions it retains more than 40% of its initial 5. The enzyme binds RBlue 5 with the highest affinity activity (data not shown). (Km  =  24  μM) of all the substrates studied. The affinity to this substrate is comparable with the affinity of DyP- Influence of hydrogen peroxide on enzyme activity type peroxidase from B. adusta (54  μM) and A. auric- With ABTS as substrate the peroxidase activity dropped ula-judae (23  μM) (Kim and Shoda 1999a; Liers et  al. significantly if the H O concentration rose above 2010). Although the binding affinity to the dye RBlue 5 2 2 0.125 mM. Inhibition of peroxidase activity by an excess by rPsaDyP is comparable, the decolorization rate of 5 −1 −1 of hydrogen peroxide via suicide inactivation has long 7.5 ×  10  s  M indicates a sixfold lower catalytic effi - 6 −1 −1 been known (Arnao et  al. 1990), but has not been fully ciency than for BadDyP and AauDyp (4.8 × 10  s  M 6 −1 −1 resolved for DyP-type peroxidases. Maximal activity and 5 × 10  s  M , respectively). was achieved at an H O concentration of 0.125  mM. The affinities of rPsaDyP for ABTS and unsubsti - 2 2 For the substrates Rblue 5, DMP, guaiacol and RBlack tuted phenols are lower. Nonetheless, the highest activ- 5 activity was inhibited at lower H O concentrations. ity and catalytic efficiency was found for ABTS. The 2 2 6 −1 −1 The inhibition of the enzyme activity strongly depends catalytic efficiency of rPsaDyP is 3.8  ×  10  s  M and upon enzyme and substrate concentration. Kim and is therefore comparable to the catalytic efficiency of Shoda (1999a) demonstrated that the degree of inhibi- the DyP-type peroxidase from Irpex lacteus (IlaDyP) 6 −1 −1 tion varied greatly depending on the substrate used. In (8.0 × 10  s  M ; Salvachúa et al. 2013) and lower than 7 −1 −1 previously described inhibition pathways Compound that of AauDyP (1.8 × 10  s  M ; Liers et al. 2010) and 7 −1 −1 II plays an important role, however, the existence of MscDyP (6.3  ×  10  s  M ; Szweda et  al. 2013). The Compound II has not been confirmed in DyP-type per - affinity of rPsaDyP is, however, also somewhat lower than oxidases (Hofrichter et  al. 2010; Sugano et  al. 2007). the affinities of AauDyP and MscDyP to ABTS. The catalytic cycle described for classical peroxidases The substituted phenols DMP and guaiacol, which in the presence of reducing substrates begins with the serve as classical substrates for MnP, are oxidized by oxidation to Compound I by the transfer of two elec- rPsaDyP. The turnover number k for the oxidation cat −1 −1 trons to H O . By the transfer of a single electron from of DMP (k   =  60  s ) or guaiacol (k   =  74  s ) are 2 2 cat cat the reduced substrate Compound II is formed, which in comparable with those of other DyP-type peroxidases −1 −1 turn is reduced to the native enzyme through reaction (AauDyP: k   =  90  s and IlaDyP: k   =  70  s ; Liers cat cat with an additional substrate molecule. In the presence et  al. 2010; Salvachúa et  al. 2013) and manganese per- −1 of excess H O Compound II reacts with H O to form oxidase (Bad MnP for DMP: k   =  70  s ; Wang et  al. 2 2 2 2 cat Compound III (inactive form). This does not necessarily 2002) and higher than those for lignin peroxidases (DMP: −1 −1 imply a final exclusion of the enzyme from the catalytic k   =  27  s , guaiacol: k   =  38  s ; Ward et  al. 2003) cat cat cycle. In the case of horseradish peroxidase there are and the versatile peroxidases (for the oxidation of DMP −1 indications that the enzyme slowly returns to the initial without Mn(II): VP from Pleurotus eryngii: k  = 3 s or cat −1 state through spontaneous decay of Compound III, giv- BadVP: k   =  2.3  s ; Camarero et  al. 1999; Mester and cat ing rise to a superoxide. Furthermore, Compound III can Field 1998). The K -values for rPsaDyP are relatively high be reduced to Compound I by various electron donors, compared to DyP–type peroxidases so that catalytic effi - allowing it to re-enter the catalytic cycle (Dequaire et al. ciency is about a fold lower. 2002). Koduri and Tien (1995) showed that the substrate Rblack 5 is a dye and a specific substrate for VP (Cama - guaiacol or the phenoxyl radical were only partially able rero et al. 1999; Heinfling et al. 1998). Liers et al. (2013b) to transform Compound III to the initial state and are showed that a number of DyP-type peroxidases oxidize considerably less efficient at this process than veratryl Rblack 5. rPsaDyP oxidizes RBlack 5 at low efficiency −1 alcohol. Dequaire et  al. (2002) presented similar results (0.1 U mg ). Lauber et al. AMB Expr (2017) 7:164 Page 13 of 15 Authors’ contributions Degradation of β-carotene and annatto (a dye mix- CL performed the experimental work on enzyme purification and its bio - ture of the xanthophylls bixin und norbixin) was chemical characterization and wrote the manuscript, TS, QKN, and PL: were demonstrated using the purified enzyme. An aqueous- responsible for the cloning and heterologous expression of the peroxidase, GL performed the Edman degradation, and HZ designed the study. All authors alkaline extract, the principle component of which was read and approved the final manuscript. the sodium salt of norbixin, was used for the oxidation of annatto (Scotter et al. 1998). The degradation of bixin Author details Institute of Food Chemistry and Food Biotechnology, Justus Liebig University was also used to determine whether rPsaDyP can oxi- Giessen, Heinrich-Buff-Ring 17, 35392 Giessen, Germany. AB Enzymes GmbH, dize both xanthophylls. The enzyme also oxidizes bixin. Feldbergstrasse 78, 64293 Darmstadt, Germany. Institute of Biochemistry, It should be noted that higher activities were determined Justus Liebig University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany. Bioresources Project Group, Fraunhofer Institute for Molecular Biology for norbixin (as an aqueous-alkaline annatto extract) at and Applied Ecology (IME), Winchesterstrasse 2, 35394 Giessen, Germany. lower substrate concentrations (15–19 μM). A compara- ble activity was measured for the oxidation of the hydro- Acknowledgements Assistance by Bruce Boschek in drafting of the manuscript is gratefully phobic substrates bixin and and β-carotene at the same acknowledged. substrate concentrations (19 μM). rPsaDyP can oxidize β-carotene without addition of Competing interests The authors declare that they have no competing interests. H O , however enzyme activity was enhanced by add- 2 2 ing H O . Enrichment of the reaction buffer with O also 2 2 2 Availability of data and materials increased the transformation of β-carotene, and con- We conducted experiments and data were generated. All data is shown in Figures and Tables within the article. versely, degassing the buffer slowed down the reaction. This indicates that the decomposition of β -carotene is Consent for publication directly dependent upon the concentration of molecular Not applicable. oxygen in the buffer. In addition, this implies that the in Ethics approval and consent to participate addition to the peroxidase function there is an oxidase- or Not applicable. oxygenase function (Sugano 2009). Zorn et al. (2003a) also Funding showed that β-carotene is degraded in an oxygen-depend- We wish to express our gratitude to the Deutschen Bundesstiftung Umwelt ent reaction by a cell-free supernatant of a Mycetinis (DBU) for financial support of the research Project (AZ 13211-32). HZ was scorodonius culture. In other studies Scheibner (2006) and financially supported by the excellence initiative of the Hessian Ministry of Science and Art which encompasses a generous grant for the LOEWE centre Hülsdau (2007) showed that the oxidation of β-carotene “Insect Biotechnology and Bioresources”. by the purified DyP-type peroxidase MsP1 from culture supernatant can take place without addition of H O , 2 2 Publisher’s Note whereby the enzyme activity was increased by addition of Springer Nature remains neutral with regard to jurisdictional claims in pub- H O . An H O independent reaction was described for lished maps and institutional affiliations. 2 2 2 2 the oxidation of epinephrine by horseradish peroxidase Received: 27 May 2017 Accepted: 17 August 2017 or for the oxidation of Indol-3-acetic acid by plant per- oxidases (Adak et al. 1998; Gazarian et al. 1998). Here, an autocatalytic process was suggested in which superoxide radicals are formed in the presence of molecular oxygen. 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Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Biotechnology
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Abstract

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 −1 55,000 U L . 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 H O by rPsaDyP, however enzyme activity was increased by the 2 2 addition of H O . This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a 2 2 consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family. Keywords: Pleurotus sapidus, Dyp-type peroxidases, White rot, β-carotene, Anthraquinone dyes, Lignin degradation Introduction prosthetic group (Poulos 2010; Welinder 1992). The DyP- Heme peroxidases have been classified into various type peroxidases, however, show no homology to any superfamilies according to their functional and struc- other known peroxidase families. They possess a unique tural properties (Morgenstern et  al. 2008). According to characteristic that differentiates them from other heme the classification of Welinder ( 1992) DyP-type peroxi- peroxidases and thus they consequently form their own dases were assigned to Class II of the plant-peroxidase superfamily (EC 1.11.1.19) among the heme-peroxidases. superfamily. This class includes the secretory fungal per - Recently Zámocký et  al. (2009, 2015) have suggested a oxidases and is characterized by a wide homogeneity; for new classification based on the overall fold, the struc - example the manganese peroxidases (MnP), lignin perox- ture of the active center and enzymatic activity. DyP- idases (LiP) and the versatile peroxidases (VP) all belong type peroxidases are now consequently allocated to the to this class (Lundell et al. 2010; Martíınez 2002). All class peroxidase-cyclooxygenase superfamily that is character- II peroxidases are extracellular and contain heme as the ized 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 *Correspondence: holger.zorn@uni-giessen.de 1 peroxidase type was discovered by Kim et al. (1995). The Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Heinrich-Buff -Ring 17, 35392 Giessen, Germany first enzyme of this family (Bad DyP) was extracted from Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lauber et al. AMB Expr (2017) 7:164 Page 2 of 15 the fungus Bjerkandera adusta and was consequently As a result of their wide substrate spectrum DyP-type purified and characterized (Kim and Shoda 1999a). In peroxidases are interesting tools for biotechnological the meantime DyP-type peroxidases have not only been processes used in the decomposition of xenobiotic anth- discovered in Basidiomycota, but also in Ascomycetes raquinone derivatives in sewage and soil (Husain 2006; and bacteria (Hofrichter et  al. 2010). This also implies Sugano et al. 2007) and are already finding use in the food that these peroxidases have a common origin before the industry. For example, the DyP-type peroxidase MsP1 division of the domains (Sugano 2009). The classical DyP from M. scorodonius (Scheibner et al. 2008), marketed as from Bjerkandera adusta is the most completely charac- MaxiBright (DSM, Heerlen, the Netherlands), is used terized member of the family of DyP-type peroxidases. to whiten whey for cheese production. The enzyme thus The term dye decolorizing peroxidase “DyP” presently represents an alternative to chemical bleaches (Szweda describes a more polyphyletic group of enzymes (Ahmad et al. 2013). et  al. 2011). A subdivision of the peroxidases into three In the present report we publish a cDNA sequence that groups (P, I, V) has recently been suggested for the clas- codes for a DyP-type peroxidase from Pleurotus sapi- sification of DyP-type peroxidases (Yoshida and Sugano dus. Only two reports have been published on the DyPs 2015). from Pleurotus fungi (Faraco et  al. 2007; Fernández- Ten DyP-type peroxidases from fungi have been thus Fueyo et  al. 2015). The enzyme was efficiently expressed far characterized, and only those from B. adusta und A. heterologously in Trichoderma reesei, purified and bio - auricula-judae have been characterized from a structural chemically characterized. Homology studies allow the and mechanical perspective (Linde and Coscolñas 2014; identification of important catalytic amino acids and Linde et  al. 2015a; Strittmatter et  al. 2013; Sugano 2009; potential substrate oxidation sites. Yoshida et  al. 2011, 2012). DyP–type peroxidases pos- sess a unique H O -binding site that differs from those Materials and methods 2 2 of other peroxidases (Sugano et al. 1999). Whereas in the Chemicals enzymes of the superfamily of plant peroxidases the rear- Chemicals and reagents used were obtained from Carl rangement of the protons of H O is typically mediated Roth (Karlsruhe, Germany), Sigma (Neu-Ulm, Germany) 2 2 by the distal histidine this is accomplished by an aspar- or Merck (Darmstadt, Germany). Chemicals and materi- tate in DyP-type peroxidases (Sugano et al. 2007). als for electrophoresis were from Serva and Bio–Rad. DyP-type peroxidases are catalytically very versatile and stable (Pühse et al. 2009). They possess a high redox Strains and culture methods potential (1.1–1.2  V) so that numerous substrates can Pleurotus sapidus (DSM 8266) was obtained from the serve as electron donors (Liers et  al. 2013a). DyP-type German Collection of Microorganisms and Cell Cultures peroxidases are therefore able to oxidize a wide spectrum (DSMZ, Braunschweig, Germany). The Basidiomycete −1 of complex dyes, in particular xenobiotic anthraquinone was cultivated in standard culture medium (30 g L d– −1 −1 derivatives as well as typical peroxidase substrates such (+)–glucose·H O, 4.5  g  L l-asparagine·H O, 1.5  g  L 2 2 −1 −1 as ABTS (2,2′-azino-bis(3-ethylthiazoline-6-sulfonate)) KH PO , 0.5  g  L MgSO ·H O, 3.0  g  L yeast extract, 2 4 4 2 −1 −1 and phenols (Sugano 2009). Certain Dyp-type peroxi- 1 mL L trace element solution: 5 mg L CuSO ·5H O, 4 2 −1 −1 dases cleave β-carotene and other carotenoids, non-phe- 80  mg  L FeCl ·6H O, 90  mg  L ZnSO ·7H O, 3 2 4 2 −1 −1 nolic bonds such as veratryl alcohol and the β-O-4 lignin 30 mg L MnSO ·H O, and 0.4 g L EDTA; pH 6.0) for 4 2 model dimer adlerol (Liers et al. 2010). 10 days (24 °C, 150 rpm, 25 mm shaking diameter). Pleurotus sapidus is a Basidiomycete of the family Pleu- Escherichia coli (TOP10) was obtained from Invitrogen rotaceae (oyster mushrooms) belonging to the white-rot (Karlsruhe, Germany) and was used for vector propa- fungi. It grows on wood (pillar fungus). White-rot fungi gation. TSS competent cells were transformed by heat are the most efficient lignin destruents and are able to shock treatment (2  min, 42  °C) according to the stand- fully decompose complex polymers (Rajarathnam et  al. ard protocols. Recombinant cells were cultivated in ster- −1 −1 1998). In addition, P. sapidus is able to decompose natu- ile LB medium (10 g L tryptone, 5 g L yeast extract, −1 −1 ral pigments such as β-carotene (Schüttmann et al. 2014). 10 g L NaCl) with 150 mg L ampicillin used as selec- Numerous enzymes that are involved in lignocellulose tion marker (37 °C, 225 rpm). decomposition have been identified in the secretome of P. sapidus. For example, the fungus secretes cellulases, cDNA‑synthesis hemicellulases, peptidases, esterases, laccases and in par- To isolate the RNA the mycelium of a P. sapidus sub- ticular peroxidases into the culture medium (Zorn et  al. mersion culture was harvested on culture day 5 and 100 2005). mg was disintegrated by grinding in liquid nitrogen. Lauber et al. AMB Expr (2017) 7:164 Page 3 of 15 TM 2, BioTek Instruments GmbH, Bad Friedrichshall, Isolation of the total RNA was performed with RNeasy Germany) or alternatively in a UV/Vis spectrophotom- Plant Mini Kits (Qiagen, Hilden, Germany) according to eter (SPECORD 50, Analytik Jena AG, Jena, Germany). the manufacturer’s instructions. The quality of the RNA Reactions were initiated by addition of the enzyme. was verified by agarose electrophoresis and ethidium Enzyme activity was measured over a period of 10  min bromide staining. A cDNA library of P. sapidus was pro- at 25  °C at the appropriate wavelength for the substrate. duced using the isolated RNA as template. cDNA syn- One unit (1 U) is defined as the amount of enzyme that thesis was performed with help of the SMART PCR converts 1  µmol substrate per minute. Various H O cDNA Synthesis Kits (Clontech Laboratories Inc., Saint- 2 2 concentrations (0–1250  µM, enzyme concentrations Germain-en-Laye, France). SuperScript   II Reverse (0.27–54 nM) and substrate concentrations were used to Transcriptase (Invitrogen, Darmstadt, Germany) was determine the enzyme activity. used for first-strand synthesis. Amplification of a cod - The activity of rPsaDyP vs ABTS was determined in ing DyP-type peroxidase sequence of P. sapidus from 100 mM sodium acetate buffer at pH 3.8 and a final H O the cDNA library was performed by PCR. The prim - 2 2 concentration of 125 µM. Production of the ABTS cation ers DTP_for58 (5′-ATGCGCTGGTGGACTACC-3′) radical was studied according to Eggert et  al. (1996) at and DTP_rev54 (5′-TTAAGCAGCGATTTTGTGC-3′) −1 −1 420 nm (ε 36,000 L mol  cm ). were derived from a homologous Dyp-type peroxidase Oxidation of DMP and guaiacol was followed in sequence from the genome (DOE-JGI) of Pleurotus 50  mM sodium acetate buffer at pH 4.5 at a final H O ostreatus. Amplification of the specific cDNA occurred 2 2 concentration of 62.5  µM as based on the forma- in an Alpha SC PCR Thermocycler (Analytik Jena, Jena, tion of the dimeric quinone derivate at 469  nm (ε Germany). The following PCR protocol was followed: −1 −1 27,500  L  mol   cm referred to the substrate) accord- 50  ng template, 5×  PCR-Puffer including dNTP’s (Qia - ing to Saparrat et  al. (2002) or tetraguaiacol at 470  nm gen), forward and reverse primer 50  pmol, 1.25  U Hot- −1 −1 (ε 26,600 L mol  cm ) according to Koduri and Tien Star HiFidelity DNA-Polymerase (Qiagen) ddH O ad 2 470 (1995). 50  µL, 95  °C 5  min–95  °C 1  min, 51  °C 1  min, 72  °C Oxidation of Reactive Blue  5 dye (Rblue 5) was per- 2 min, 40 cycles–72 °C 5 min. PCR products were sepa- formed in 100  mM sodium acetate buffer at pH 4.0 rated electrophoretically (1.2% (w/v) agarose gel), subse- and a final H O concentration of 31.2  µM. Degrada- quently isolated from the gel using NucleoSpin Extract 2 2 tion of the substrate was determined at 600  nm (ε II–Kits (Machery-Nagel, Düren, Germany) and finally −1 −1 8000 L mol  cm ) according to Sugano et al. (2006). ligated (Topo TA-Cloning Kit, Invitrogen) in the vec- Oxidation of the azo dye Reactive Black 5 (RBlack 5) tor pCR2.1–TOPO (Invitrogen). The plasmid DNA was was determined in 50 mM sodium acetate buffer at pH 4.0 replicated in E.  coli TOP10 cells (Invitrogen), isolated −1 −1 with 62.5 µM H O at 598 nm (ε 37,200 L mol  cm ) from the cells and purified (NucleoSpin Plasmid DNA 2 2 598 according to Sugano et al. (2006). Purification, Machery-Nagel). Sequencing of the cloned A β-carotene stock solution was prepared as described cDNA was performed by MWG Eurofins (Ebersberg, by Pühse et  al. (2009). Measurement of the substrate Germany). degradation was performed at 450  nm (ε 95,000 −1 −1 L  mol   cm ) according to Ben Aziz et  al. (1971) in Enzyme production 50 mM sodium acetate buffer at pH 3.5 with a final H O For recombinant production of the peroxidase the codon 2 2 concentration of 125 µM. To assess the purified enzyme’s usage of the gene was adapted to the host organism oxygenase activity in the absence of H O , 100  mL Trichoderma reesei (a derivative of RUT C30; Peterson 2 2 sodium acetate buffer (100 mM, pH 3.8) was purged with and Nevalainen 2012) and an expression cassette was oxygen through a glass frit (pore size 3, pore diameter constructed from the adapted sequence. The expression 16–40  μm) for 30  min. For control experiments in the of peroxidase took place under the control of the cbhl absence of H O and oxygen, 100  mL of the buffer was promotor and terminator. Efficient production of the 2 2 sonicated for 60  min, and residual oxygen was removed peroxidase was achieved by optimization of the fermen- by purging with nitrogen. The assays were performed tation process under fed-batch conditions (pH5.5, 28 °C, with enzyme concentrations of 270, 360, and 540  nM, 160 h) in Monosaccharide medium (7% monosaccharide, and the concentration of the substrate was 26 µM. 4% agricultural waste stream derived N-source, 0.7% As further examples of natural dyes annatto (aque- (NH ) SO , 0.3% KH PO ). 4 2 4 2 4 ous alkaline extract), the principle component of which is norbixin, and bixin were used as substrates. Prepara- Determination of enzyme activity tion of the bixin stock solution was analogous to that of Enzyme activity was determined photometrically using a β-carotene (using 15  mg bixin). Conversions took place temperature controlled multi-mode plate reader (Synergy Lauber et al. AMB Expr (2017) 7:164 Page 4 of 15 in 50  mM sodium acetate buffer at pH 6.0 or 3.5 with a of the proteins took place in a 6% stacking and a 12% sep- final H O concentration of 125  µM. Degradation of the aration gel. Protein bands were visualized using colloidal 2 2 −1 −1 dye was observed at 452 nm (ε 108,400 L mol  cm , Coomassie staining (Kang et al. 2002). Size determination −1 −1 ™ Scotter et al. 1998) or 465 nm (ε 136,100 L mol  cm , was performed with the help of PageRuler Unstained Hülsdau 2007). Proteinladder (Fermentas, St. Leon–Rot, Germany). For Samples of heat inactivated (95  °C, 10  min) enzyme N-deglycosylation samples were incubated with PNGase served as negative controls. F (NEB, Ipswich, USA) and treated following the manu- facturer’s instructions. Purification of the DyP‑type peroxidase by fast protein The size of the native proteins was determined by gel liquid chromatography (FPLC) filtration chromatography (GFC) and Blue Native PAGE. Purification was carried out in a cold room at 4 °C. Chro - For GFC the protein was loaded onto a Superdex 200 matographic separation of the proteins was performed 10/300 GL column (GE Healthcare) and eluted with using a BioLogic DuoFlow (Biorad) FPLC–System with a 50  mM sodium acetate buffer pH 4.0 with 250  mM fraction collector (BioLogic BioFrac fraction collector, NaCl and 0.005% Triton X-100 at a flow rate of 0.5  mL −1 Biorad). Proteins were detected at 280 nm. The DyP-type min . For Blue Native PAGE precast gels from Serva peroxidase was purified in a two-stage process by hydro - (SERVAGel N Native Starter Kit, Serva, Heidelberg, phobic interaction chromatography and ion exchange Germany) and the PerfectBlue Double Gel System Twin chromatography. The culture supernatant was concen - S of Peqlab were used. For isoelectric focusing (IEF) the trated (Macrosep , 10  kDa cutoff, Pall, Dreieich, Ger - same electrophoresis system was used (precast vertical many) and transferred to 50  mM sodium acetate buffer gels and IEF marker 3–10, Liquid Mix, Serva). Visuali- at pH 4.0 with 1 M (N H ) SO . In the first stage the con - zation of the protein bands was performed as described 4 2 4 centrated culture supernatant was purified on a column above. In addition, specific staining of heme- and metal with phenyl-Sepharose-matrix (HiPrep Phenyl FF (high enzymes was performed with 3,3′,5,5′-tetramethylb- −1 sub) 16/10, GE Healthcare Bio–Sciences AB, Uppsala, enzidine (TMB) (1.5  mg  mL in methanol) modi- Sweden). The Starting buffer used was 50  mM sodium fied according to Thomas et  al. (1976) and Henne et  al. acetate buffer pH 4.0 with 1  M (NH ) SO and proteins (2001). Native electrophoresis was followed by activ- 4 2 4 were eluted over a gradient of 0 to 58% (40  mL) and ity staining with ABTS as substrate. For this the gel was from 58 to 100% (30  mL) sodium acetate buffer pH 4.0. equilibrated for 5  min in 100  mL freshly prepared stain −1 Flow rate was 3  mL  min and fractions with a volume solution (5 mM ABTS in 50 mM sodium acetate pH 4.0) of 2 mL were collected. Protein containing fractions were on an orbital shaker. Subsequently, 150 μL 3% H O (final 2 2 tested and active fractions pooled for the second purifi - concentration 130  μM) was added and shaking contin- cation step. They were then concentrated, transferred to ued until a distinct staining was obtained. Gels were then 50  mM sodium acetate buffer pH 4.0 and loaded onto a rinsed with pure water and documented. column with SP Sepharose Fast Flow Matrix (XK16/20, 25 mL column volume, GE Healthcare Bio–Sciences AB). Influence of pH value Elution was performed on a gradient of 0–10% (30  mL) The optimum pH of the purified rPsaDyP was deter - and from 10 to 100% (30  mL) 50  mM sodium acetate mined in McIlvaine–Puffer (pH 2.2–7.5, McIlvaine, −1 buffer pH 4.0 with 1 M NaCl at a flow rate of 3 mL min . 1921), 50  mM sodium acetate buffer (pH 3.0–6.0) and Active fractions of the second purification stage were 100 mM sodium tartrate buffer (pH 2.0–5.5) with ABTS pooled, concentrated, aliquoted, shock frozen in liquid as substrate. The pH stability was determined by dilut - nitrogen and stored at −80 °C. ing the DyP-type peroxidase in sodium acetate buffer at a pH range of 3.0–6.0 (0.5 steps). To avoid altering the Enzyme characterization pH value too drastically for the measurement the sam- Protein concentration was determined according to ples were again diluted 1:10 with 100 mM sodium acetate Bradford (1976) using the dye solution Roti Nanoquant buffer pH 3.8 (measurement buffer). Rest activity was (Roth, Karlsruhe, Germany) with bovine serum albumin measured after 0.5, 1, 2 and 24  h storage at 4  °C using or lyophilized DyP as standard. ABTS as substrate and values were compared to the ini- UV/Vis spectra of the purified rPsaDyP were recorded tial activity. at 250 nm to 800 nm in a NanoPhotometer (Pearl, Implen GmbH, Munich, Deutschland). Eec ff t of temperature Molecular mass determination of the DyP-type peroxi- To determine the optimum pH for the purified rPsaDyP dase was performed using SDS–PAGE (Mini–Protean the enzyme was initially incubated for 4  min in 50  mM TetraSystem, BioRad) according to Laemmli. Separation sodium acetate buffer at pH 3.8 at various temperatures Lauber et al. AMB Expr (2017) 7:164 Page 5 of 15 (15–75  °C). The rate of turnover was then measured at Structure and sequence based analysis the corresponding temperature in a photometer with A structural homology model of the DyP-type peroxidase temperature regulated cuvette. from P. sapidus was calculated based on the x-ray crystal structure of DyP-type peroxidase from A. auricula-judae Determination of kinetic parameters (PDB-ID 4AU9B). The structures exhibit a homology of The apparent value of the Michaelis–Menten constant 44%. (K ) and catalytic constant (k ) of the purified rPsaDyP The calculated structural model possesses a helical m cat were determined for ABTS, DMP, guaiacol and RBlue 5. basic structure with two domains (proximal N-termi- Substrate turnover took place at 30 °C in 6 replicate prep- nal, distal C-terminal domains), that enclose the central arations. The initial rate was determined with constant cofactor heme. A prominent motif made up of anti-par- enzyme concentration, varying substrate concentrations allel β-sheet structures that, together with the α-helices, and constant cosubstrate concentration. Measurements exhibit a ferredoxin-like folding is present on the distal were taken in parallel with varying H O concentrations side of the heme. With the help of this structure homol- 2 2 in order to determine the H O concentration that no ogy model it was possible to identify important func- 2 2 longer had a limiting effect on the activity. The follow - tional amino acid residues (Fig.  2). The access channel ing substrate concentrations were chosen for determi- to the heme of the catalytic channel forms a predomi- nation of the kinetic parameters: ABTS 15–1500  µM, nantly hydrophobic channel through which the sub- DMP 250–15,000 µM, guaiacol 500–15,000 µM, RBlue 5 strate reaches the H O binding pocket (Yoshida et  al. 2 2 11–300 µM. The initial rate (ν ) was plotted linear to the 2011). Three of the remaining residues of the H O bind- 0 2 2 substrate concentration in a graph (Cornish-Bowden ing pocket are also conserved in rPsaDyP, whereby in diagram). Using the software Origin (OriginPro 8.6G) a the DyP-type peroxidases from P. sapidus and P. ostrea- saturation hyperbola was adapted through a non-linear tus leucine is exchanged for valine (rPsaDyPV363). regression in order to obtain values for K and ν . The The catalytic residues of D174—part of the conserved m max catalytic constant k was determined using the following GxxDG motif—and R338 on the proximal side H317 as cat equation, whereby E represents the enzyme concentra- fifth ligand of heme were identified. These residues are tion applied: involved in the activation of the enzyme (formation of compound I) by heterolytic cleavage of H O . Based on 2 2 max k = the alignment (Fig.  2) it was shown that E391, which cat [E] forms a hydrogen bond with the fifth ligand of heme Results (H317), thus stabilizing compound I (Sugano et al. 2007), Identification of the coding sequence of DyP‑type is exchanged for an aspartate (rPsaDyP D401) in other peroxidase Dyp-type peroxidases. The arginine 267 and 324 hydro - The 1551 bp full-length cDNA of a DyP-type peroxidase gen bonds with the propionate residues of the heme gene of Pleurotus sapidus (strain 8266) was amplified. form additional ligands. The conserved amino acid resi - This gene codes for a protein of 516 AA (Fig.  1, Acces- dues Y343 and W383 that were exposed to the solvent sion Number LN830264), a theoretical molecular weight are found on the surface. These serve as oxidation sites of 57.1  kDa and a calculated isoelectric point (pl) of for large substrates and may be elements of a LRET (long 5.73 (ExPASy ProtParam). The ORF exhibits 4 potential range electron transfer) (Liers et  al. 2013a; Strittmatter N-glycosylation sites and a potential O-glycosylation site et al. 2013). (NetNGly 1.0, NetOGly3.1). With the identification of putative conserved domains Heterologous expression and purification the enzyme is assigned to the dye decolorizing peroxi- For purification and characterization peroxidase positive dase superfamily (EMBL-EBI, InterProScan). In addition, transformants were cultivated in large scale (XL) under the structural motif GxxDG that is present in all mem- conditions that yield active protein in the culture super- −1 bers of the DyP-type peroxidase family was found in the natant. After 160 h cultivation an activity of 55,000 U L sequence. The translated amino acid sequence shows a in relation to the substrate ABTS was achieved and the high degree of identity (95%) to a DyP-type peroxidase supernatant containing the peroxidase was harvested. from Pleurotus ostreatus (ID EMBL CAK55151.1). Fur- After concentration of the culture supernatant (crude thermore, the amino acid sequence exhibits an identity enzyme) by a factor of 5 the Dyp-type peroxidase was of 42–43% with DyP-type peroxidases from B. adusta purified in a two-stage sequential procedure using FPLC (Accession Number: BAA77283.1), M. scorodonius with hydrophobic interaction chromatography and ion (Accession Number: B0BK71.1) and A. auricula-judae exchange chromatography. The DyP-type peroxidase (Accession Number: AFJ79723). was purified to apparent homogeneity. After the second Lauber et al. AMB Expr (2017) 7:164 Page 6 of 15 Fig. 1 Nucleotide- and derived amino acid sequence of a DyP-type peroxidase from Pleurotus sapidus (LN830264). Predicted N-glycosylation sites are highlighted; a hypothetical cleavage site after 62 amino acids is marked with an arrow Lauber et al. AMB Expr (2017) 7:164 Page 7 of 15 Fig. 2 Alignment of various DyP-type peroxidase sequences from P. sapidus (PsaDyP), P. ostreatus (PosDyP, NCBI: CAK55151), B. adusta (BadDyP, NCBI: BAA77283), A. auricula-judae (AauDyP, NCBI: AFJ79723) and M. scorodonius (MsP1, NCBI: BOBK71). Conserved amino acids (*); the character- istic GxxDG motif is enclosed in a frame; remnants of the H O binding site (circle); E391 (blue); conserved residues of the heme binding site (gray 2 2 background); potential heme binding residues (triangle); exposed amino acids of a potential LRET (long range electron transfer, green background); remnants involved in a potential LRET (green); N-terminal conserved amino acid sequences (white); conserved histidine residues (H164 and H166, blue background); signal sequences (gray); N-terminal amino acids are underlined Lauber et al. AMB Expr (2017) 7:164 Page 8 of 15 purification step the active fractions eluted in a single, oligosaccharides from the N-glycosylation sites. After discreet peak (Fig.  3). Following the two-stage puri- treatment with the glycosidase rPsaDyP showed a lower fication an electrophoretic homogeneity of DyP was apparent molecular weight in SDS-PAGE than the −1 achieved with a yield of 2.9 ± 0.1 g L and an increase in untreated samples (52.8–57.6  kDa). This indicates that Reinheitszahl from 0.2 to 1.1. rPsaDyP is glycosylated by the host organism. From the difference in molecular weights a degree of glycosylation Characterization of rPsaDyP of ~9% was calculated. The purified enzyme showed a typical heme-enzyme red coloring and a maximum absorbance λ  = 409 nm and N‑terminus max two further maxima at 510 and 640 nm (Fig. 4). A molec- The detected apparent molecular weight of the deglyco - ular weight of 57.6  kDa was determined by SDS-PAGE. sylated rPsaDyP is lower than the theoretical weight that Isoelectric focusing (IEF) showed a pl of 6.7 (Fig. 4). was calculated from the primary sequence. This suggests that the protein is processed in the host organism. There - Determination of the native conformation fore, the N-terminus of the recombinant enzyme was The native conformation of the DyP was established by determined by Edman degradation. The first 12 residues Blue Native PAGE and gel filtration chromatography. of the N-terminal amino acid sequence of the purified By electrophoresis a molecular weight of 115  kDa was enzyme (AT(N)GTFLPLEEI) were identified. According ascertained for the native, active enzyme and the reten- to this the enzyme has a signal peptide 62 amino acids tion time of the peaks in gel filtration chromatography long and the mature protein a total length of 454 amino indicated an apparent molecular weight of 122 kDa. The acids. molecular weights indicated by these two methods were comparable and approximately double the value of the Catalytic properties of rPsaDyP apparent molecular weight of rPsaDyP under denaturing pH‑ and temperature optimum conditions (57.6  kDa). This suggests that the quaternary Before the kinetic constants were determined the optimal structure of the native enzyme is a dimer. pH for oxidation of the substrate investigated was identi- Four potential N-glycosylation sites were predicted fied (Fig.  5). The pH-optimum for the oxidation of ABTS in the amino acid sequence. The protein was therefore and Rblue 5 was found to be between 3.5 and 4.0, for the treated with PNGase F in order to remove any possible reaction with phenolic substrates the pH-optimum was Fig. 3 Cation-exchange chromatography as second purification step using an SP Sepharose FF column; ABTS oxidizing enzyme activity was detected in the area with a gray background; UV absorption in AU at 28 nm (blue line), detected activity (dashed line), buffer concentration (50 mM sodium acetate buffer pH 4.0 with 1 M NaCl) in % (light gray line) Lauber et al. AMB Expr (2017) 7:164 Page 9 of 15 Fig. 4 a UV–Vis absorption spectrum of the purified rPsaDyP showing the Soret band at 409 nm and two additional maxima in the region of 510 and 640 nm; b isoelectric focusing of purified rPsaDyP, stained with colloidal Coomassie (1) and specific staining for heme/metal enzymes with TMB (2); M IEF Marker 3–10 Fig. 5 a Optimum pH value for oxidation of ABTS, DMP and guaiacol by rPsaDyP in 50 mM sodium acetate buffer or Rblue 5 in 100 mM sodium tartrate buffer. b Eec ff t of temperature on the activity of rPsaDyP with ABTS as substrate higher (pH 4.5). In addition, the enzyme activity for the stability residual activity was detected after a 5 min incu- substrates was determined under varying H O concentra- bation of the enzyme. At temperatures over 70  °C the 2 2 tions (0–1250 µM). With ABTS as substrate the peroxidase enzyme completely lost activity. A 5–min T –Wert of activity fell significantly when the H O concentration rose 53  °C was determined from the residual activity of the 2 2 above 0.125 mM, indicating that the enzyme is inhibited by enzyme. Under assay conditions the enzyme showed a H O . Maximum reaction rates depending upon substrate 75% residual activity after 2  h and after 24  h 40% of the 2 2 tested reached values between 31.2 and 125 µM (31.2 µM: initial activity was still present. RBlue 5, 62.5  µM: DMP, guaiacol, RBlack 5; ABTS, Ann- atto, Bixin, β-carotene: 125 µM). Catalytic properties The enzyme showed maximum activity over a tem - The apparent kinetic constant for rPsaDyP (expressed perature range of 15–30  °C. To determine the thermal in T. reesei as active protein) was determined for ABTS, Lauber et al. AMB Expr (2017) 7:164 Page 10 of 15 RBlue 5, DMP and guaiacol as shown in Table 1. The per - Table 2 Degradation of  β-carotene by  the purified recom- binant DyP-type peroxidase from Pleurotus sapidus (in the oxidase efficiently oxidized the low redox potential dye absence of  H O ) under standard assay conditions, in oxy- ABTS. In addition, rPsaDyP catalyzed the degradation 2 2 gen saturated buffer, and under anoxic conditions of the dye Rblue5, a characteristic DyP substrate. The −1 enzyme binds this substrate at high affinity (K  = 24 μM) Enzyme concen‑ v (µM min ) tration (nM) and converts it efficiently. The substrate spectrum of Degassed buffer Standard assay Oxygen rPsaDyP also includes the substituted phenols DMP and conditions saturated buffer guaiacol. Both of these substrates show a high K (713 and 1227  µM, respectively) and a relatively low catalytic 270 0.045 0.385 0.878 efficiency compared to the other substrates. 360 0.056 0.495 1.069 DyP is also able to catalyze the degradation of the nat- 540 0.077 0.532 1.217 −1 ural pigment β-carotene ene (90  U  L ). A study of the kinetics of the conversion of this substrate was difficult, however, since only low substrate concentrations (maxi- mal 26  µM) could be used in the assay and thus satura- was cloned and sequenced. By comparing the converted tion of the enzyme could not be achieved. Notably, the amino acid sequence with other DyP-type peroxidases, DyP-type peroxidase can convert this substrate without and with the structural homology model of the structure the addition of H O in the same manner as MsP1 and of AauDyP as a basis, shared motifs and catalytic residues 2 2 MsP2 (Scheibner et  al. 2008; Zorn et  al. 2003b). At the could be identified. The model demonstrates the charac - same time, enrichment of the buffer with O increased teristic β-barrel structure and environment of the heme the conversion rate by a factor of 2.3 compared to stand- pocket. This includes the characteristic GxxDG motif ard conditions without addition of H O . The enzymatic with the conserved aspartate that, together with the con- 2 2 conversion was virtually halted when degassed buffer was served arginine residue is situated on the distal side of the used (Table  2). This indicates that the enzyme also pos - heme, as well as the proximal histidine. The alignment sesses oxidase or oxygenase activity. shows that the amino acids involved in the heme binding Furthermore, the enzyme can also convert additional as well as those involved in catalysis are highly conserved. natural pigments such as bixin and annatto (90 and DyP-type peroxidases are also capable of oxidizing −1 114  U  L ), as well as high redox potential dyes such as large substrates that are not able to reach the immediate −1 RBlack 5 (231 U L ). proximity of the heme in the active center. For this reason it is a matter of discussion as to whether these enzymes Discussion exclusively possess solvent-exposed substrate binding The first published sequence of a DyP-type peroxidase sites (Liers et al. 2013a). Strittmatter et al. (2013) identi- originated from the Basidiomycetes B. adusta (Kim and fied potential LRET transfer pathways in the DyP-type Shoda 1999b). In the meantime further DyP-type per- peroxidase from A. auricula-judae. A number of exposed oxidases from white-rot fungi have been cloned and residues on the protein surface (Trp or Tyr) serve as sequenced, including P. ostreatus, M. scorodonius und A. oxidation site for large substrates. Linde et  al. (2015a, auricula–judae (Faraco et al. 2007; Scheibner et al. 2008; b) recently showed that LRET from AauDyP essentially Liers et al. 2010). X-ray structural analysis has only been begins at W377 that is also conserved in rPsaDyP. performed on two DyP-type peroxidases from fungi. The histidine 164 or 166 (BadDyP) are conserved in The first DyP-peroxidase structure examined was from many DyP sequences. It was therefore long a matter of B. adusta (BadDyP; PDB-Code 2D3Q). In the meantime discussion whether these residues function as proximal the structure of a DyP from A. auricula-judae (AauDyP; histidine or as heme ligand. Sugano et al. (2004) showed PDB-Code 4AU9) has also been elucidated. In the pre- that H166 is not essential for the peroxidase activ- sent study the first DyP-type peroxidase from P. sapidus ity. On the other hand, the authors showed that mutant Table 1 Apparent kinetic constants of the recombinant DyP-type peroxidase from Pleurotus sapidus −1 −1 −1 −1 −1 Substrate pH Enzyme concentration (nM) K (µM) k (s ) k K (s  M ) v (µM s ) m cat cat m max ABTS 3.8 0.27 99 375 3.8 × 10 0.10 DMP 4.5 1.8 1227 60 4.9 × 10 0.11 Guaiacol 4.5 4.5 713 74 1.0 × 10 0.35 RBlue 5 4.0 5.4 24 18 7.5 × 10 0.10 Lauber et al. AMB Expr (2017) 7:164 Page 11 of 15 H164A completely lost the activity. This result indicates of the DyP-type peroxidase MsP2 (Scheibner et al. 2008). that H164 is not directly involved in heme binding, but The carbohydrate content of DyP-type peroxidases lies rather suggests only a decrease in protein stability and typically between 9 and 31% (Hofrichter et al. 2010). a loss of the heme-binding affinity (Faraco et  al. 2007). After deglycosylation the apparent molecular weight H164 is not conserved in rPsaDyP or in PosDyP, but is determined in SDS-PAGE was lower for rPsaDyP het- replaced by a lysine (K167). This exchange is also found erologously expressed in T. reesei than was calculated in various other representatives of the DyP-type per- from the primary sequence. This implies that the enzyme, oxidase family, for instance in the proteins of A. oryzae just as with BadDyP in A. oryzae (Sugano et  al. 2000), (Q2UPE9, Q2U1I3), Neurospora crassa (Q7S3A4) and is further processed in the host organism, T. reesei. The various other members of the DyP-family (Faraco et  al. N-terminus of the recombinant enzyme was sequenced 2007). This suggests that H164 is not directly involved in by Edman degradation. Processing of rPsaDyP is between heme binding and another residue coordinates the heme amino acids 62 and 63. Sugano et al. (2000) showed that in DyP-type peroxidases. Nonetheless, according to Sug- the recombinant DyP-type peroxidase from B. adusta ano (2009) H164 plays an important role in the folding of produced in A. oryzae has the same N-terminus as the DyP-type peroxidases and binding of heme, even if it is wild-type enzyme. This suggests that the recombinant not conserved in all members of this protein family. Here and native PsaDyP have the same N-terminus. Studies of the situation is different than described by the authors. other DyP-type peroxidases show that processing occurs Histidine does not appear to be crucial for folding, but between position 56/57 (BadDyP), 55/56 (MsP1), 57/58 rather, a basic amino acid in this position. Johjima et  al. (MsP2) and 61/62 (AauDyP) (Liers et  al. 2010; Scheib- (2003) identified 10 potential ligands (His, Tyr und Cys) ner et  al. 2008; Sugano et  al. 2000). In rPsaDyP a clus- for heme. For AauDyp Strittmatter et  al. (2013) showed ter of conserved amino acids is present in the region of that on the proximal side arginine 255 and 311 form the N-terminus, however a typical cleavage site was not hydrogen bonds to the propionate residues of heme and found (Liers et al. 2010). are involved in the coordination of heme. The homolo - In contrast to the classical DyP rPsaDyP occurs as gous residues from rPsaDyP were identified (at position a dimer, whereby it must be noted that various other 267 and 324) and in the model are at a distance of 3.2 and DyP-type peroxidases, especially from prokaryotes, 3.5 Å, respectively, to the propionates. form numerous higher quaternary structures ranging from monomers to hexamers. The reason for this oli - Heterologous expression gomerization has been subject of discussion, but remains The DyP-type peroxidase from Pleurotus sapidus was unknown. Sugano (2009) showed that the classical DyP, successfully expressed heterologously in the ascomy- compared to DyP-type peroxidases that form oligomers, cete T. reesei and the active enzyme was secreted into exhibit insertions in the primary sequence that are miss- −1 the culture supernatant. An activity of 55,000  U  L ing in the former. Nonetheless, the primary sequences was determined for the recombinant DyP-type peroxi- of MsP1 and MsP2 exhibit a high degree of homology to dase in the supernatant with the substrate ABTS. Het- BadDyP and like rPsaDyP and have these insertions, and erologous expression of the DyP from B. adusta in A. yet they occur natively as dimers (Scheibner et al. 2008). oryzae imparted a 42 fold increase in activity with the 2 −1 substrate RBlue 5 (8 × 10  U L ) over that in the culture Biochemical characterization medium (Sugano et al. 2000). Heterologous expression of DyP-type peroxidases are typically secreted glycoproteins the PsaDyP from T. reesei led to an order of magnitude with molecular weights of 40–67  kDa (in monomeric 3 −1 higher activity (5  ×  10  U L ) with RBlue 5. PsaDyP form) and isoelectric points in the acid range (3.5–4.3, could be efficiently expressed in T. reesei and unusu - Hofrichter et  al. 2010). A monomer of rPsaDyP has a ally high activities could be achieved. The recombinant molecular weight of 57.4 kDa and therefore is of average enzyme showed the characteristic absorption maximum size for an enzyme of the family of DyP-type peroxidases. at 409 nm (Soret–Band) and two further maxima (α and In contrast to other enzymes of this family rPsaDyP has β) in the region of 640 nm that are attributed to the por- an apparent Pl within the neutral range. Lignolytic per- phyrin structure of heme (Glenn and Gold 1985; Renga- oxidases from fungi (LiP, VP, DyP), like plant peroxi- nathan and Gold 1986). dases demonstrate maximum activity in an acidic milieu (pH 1.5–5.0, Camarero et  al. 1999; Gazarian et  al. 1996; Native conformation Liers et al. 2010; McEldoon et al. 1995). Depending upon A glycosylation degree of 9% was determined for the the substrate, the pH profile of rPsaDyP shows an activ - DyP-type peroxidase heterologously expressed in T. ree- ity maximum between pH 3.5 and 4. 5 (with the excep- sei, a carbohydrate content that is comparable with that tion of Annatto at pH 6.0). Thus, the profile is shifted to Lauber et al. AMB Expr (2017) 7:164 Page 12 of 15 somewhat higher pH values compared to other DyP-type for horseradish peroxidase. Compound III was reduced peroxidases. Maximum activity of rPsaDyP was shown to Compound I by various electron donors. A similar between 15 and 30  °C. This is comparable with that of substrate dependent mechanism may explain the diver- BadDyP (Kim and Shoda 1999a). Above 35 °C activity of gent inhibition of the enzyme by hydrogen peroxide in rPsaDyP decreased continuously and  ~65% remained at rPsaDyP. 50 °C. rPsaDyP is active in a wide range of temperatures and pH values. It remains active for a number of hours in Catalytic properties sodium acetate buffer between pH 3 and pH 6 and main - From the functional standpoint the heterologously tains up to 60% of its initial activity after 24  h (data not expressed peroxidase of P. sapidus can be assigned to shown). There is a tendency for the activity of the enzyme the class of DyP-type peroxidases as based on its sub- to remain stable at higher pH values. After 24  h under strate spectrum and the efficient oxidation of RBlue reaction conditions it retains more than 40% of its initial 5. The enzyme binds RBlue 5 with the highest affinity activity (data not shown). (Km  =  24  μM) of all the substrates studied. The affinity to this substrate is comparable with the affinity of DyP- Influence of hydrogen peroxide on enzyme activity type peroxidase from B. adusta (54  μM) and A. auric- With ABTS as substrate the peroxidase activity dropped ula-judae (23  μM) (Kim and Shoda 1999a; Liers et  al. significantly if the H O concentration rose above 2010). Although the binding affinity to the dye RBlue 5 2 2 0.125 mM. Inhibition of peroxidase activity by an excess by rPsaDyP is comparable, the decolorization rate of 5 −1 −1 of hydrogen peroxide via suicide inactivation has long 7.5 ×  10  s  M indicates a sixfold lower catalytic effi - 6 −1 −1 been known (Arnao et  al. 1990), but has not been fully ciency than for BadDyP and AauDyp (4.8 × 10  s  M 6 −1 −1 resolved for DyP-type peroxidases. Maximal activity and 5 × 10  s  M , respectively). was achieved at an H O concentration of 0.125  mM. The affinities of rPsaDyP for ABTS and unsubsti - 2 2 For the substrates Rblue 5, DMP, guaiacol and RBlack tuted phenols are lower. Nonetheless, the highest activ- 5 activity was inhibited at lower H O concentrations. ity and catalytic efficiency was found for ABTS. The 2 2 6 −1 −1 The inhibition of the enzyme activity strongly depends catalytic efficiency of rPsaDyP is 3.8  ×  10  s  M and upon enzyme and substrate concentration. Kim and is therefore comparable to the catalytic efficiency of Shoda (1999a) demonstrated that the degree of inhibi- the DyP-type peroxidase from Irpex lacteus (IlaDyP) 6 −1 −1 tion varied greatly depending on the substrate used. In (8.0 × 10  s  M ; Salvachúa et al. 2013) and lower than 7 −1 −1 previously described inhibition pathways Compound that of AauDyP (1.8 × 10  s  M ; Liers et al. 2010) and 7 −1 −1 II plays an important role, however, the existence of MscDyP (6.3  ×  10  s  M ; Szweda et  al. 2013). The Compound II has not been confirmed in DyP-type per - affinity of rPsaDyP is, however, also somewhat lower than oxidases (Hofrichter et  al. 2010; Sugano et  al. 2007). the affinities of AauDyP and MscDyP to ABTS. The catalytic cycle described for classical peroxidases The substituted phenols DMP and guaiacol, which in the presence of reducing substrates begins with the serve as classical substrates for MnP, are oxidized by oxidation to Compound I by the transfer of two elec- rPsaDyP. The turnover number k for the oxidation cat −1 −1 trons to H O . By the transfer of a single electron from of DMP (k   =  60  s ) or guaiacol (k   =  74  s ) are 2 2 cat cat the reduced substrate Compound II is formed, which in comparable with those of other DyP-type peroxidases −1 −1 turn is reduced to the native enzyme through reaction (AauDyP: k   =  90  s and IlaDyP: k   =  70  s ; Liers cat cat with an additional substrate molecule. In the presence et  al. 2010; Salvachúa et  al. 2013) and manganese per- −1 of excess H O Compound II reacts with H O to form oxidase (Bad MnP for DMP: k   =  70  s ; Wang et  al. 2 2 2 2 cat Compound III (inactive form). This does not necessarily 2002) and higher than those for lignin peroxidases (DMP: −1 −1 imply a final exclusion of the enzyme from the catalytic k   =  27  s , guaiacol: k   =  38  s ; Ward et  al. 2003) cat cat cycle. In the case of horseradish peroxidase there are and the versatile peroxidases (for the oxidation of DMP −1 indications that the enzyme slowly returns to the initial without Mn(II): VP from Pleurotus eryngii: k  = 3 s or cat −1 state through spontaneous decay of Compound III, giv- BadVP: k   =  2.3  s ; Camarero et  al. 1999; Mester and cat ing rise to a superoxide. Furthermore, Compound III can Field 1998). The K -values for rPsaDyP are relatively high be reduced to Compound I by various electron donors, compared to DyP–type peroxidases so that catalytic effi - allowing it to re-enter the catalytic cycle (Dequaire et al. ciency is about a fold lower. 2002). Koduri and Tien (1995) showed that the substrate Rblack 5 is a dye and a specific substrate for VP (Cama - guaiacol or the phenoxyl radical were only partially able rero et al. 1999; Heinfling et al. 1998). Liers et al. (2013b) to transform Compound III to the initial state and are showed that a number of DyP-type peroxidases oxidize considerably less efficient at this process than veratryl Rblack 5. rPsaDyP oxidizes RBlack 5 at low efficiency −1 alcohol. Dequaire et  al. (2002) presented similar results (0.1 U mg ). Lauber et al. AMB Expr (2017) 7:164 Page 13 of 15 Authors’ contributions Degradation of β-carotene and annatto (a dye mix- CL performed the experimental work on enzyme purification and its bio - ture of the xanthophylls bixin und norbixin) was chemical characterization and wrote the manuscript, TS, QKN, and PL: were demonstrated using the purified enzyme. An aqueous- responsible for the cloning and heterologous expression of the peroxidase, GL performed the Edman degradation, and HZ designed the study. All authors alkaline extract, the principle component of which was read and approved the final manuscript. the sodium salt of norbixin, was used for the oxidation of annatto (Scotter et al. 1998). The degradation of bixin Author details Institute of Food Chemistry and Food Biotechnology, Justus Liebig University was also used to determine whether rPsaDyP can oxi- Giessen, Heinrich-Buff-Ring 17, 35392 Giessen, Germany. AB Enzymes GmbH, dize both xanthophylls. The enzyme also oxidizes bixin. Feldbergstrasse 78, 64293 Darmstadt, Germany. Institute of Biochemistry, It should be noted that higher activities were determined Justus Liebig University Giessen, Friedrichstrasse 24, 35392 Giessen, Germany. Bioresources Project Group, Fraunhofer Institute for Molecular Biology for norbixin (as an aqueous-alkaline annatto extract) at and Applied Ecology (IME), Winchesterstrasse 2, 35394 Giessen, Germany. lower substrate concentrations (15–19 μM). A compara- ble activity was measured for the oxidation of the hydro- Acknowledgements Assistance by Bruce Boschek in drafting of the manuscript is gratefully phobic substrates bixin and and β-carotene at the same acknowledged. substrate concentrations (19 μM). rPsaDyP can oxidize β-carotene without addition of Competing interests The authors declare that they have no competing interests. H O , however enzyme activity was enhanced by add- 2 2 ing H O . Enrichment of the reaction buffer with O also 2 2 2 Availability of data and materials increased the transformation of β-carotene, and con- We conducted experiments and data were generated. All data is shown in Figures and Tables within the article. versely, degassing the buffer slowed down the reaction. This indicates that the decomposition of β -carotene is Consent for publication directly dependent upon the concentration of molecular Not applicable. oxygen in the buffer. In addition, this implies that the in Ethics approval and consent to participate addition to the peroxidase function there is an oxidase- or Not applicable. oxygenase function (Sugano 2009). Zorn et al. (2003a) also Funding showed that β-carotene is degraded in an oxygen-depend- We wish to express our gratitude to the Deutschen Bundesstiftung Umwelt ent reaction by a cell-free supernatant of a Mycetinis (DBU) for financial support of the research Project (AZ 13211-32). HZ was scorodonius culture. In other studies Scheibner (2006) and financially supported by the excellence initiative of the Hessian Ministry of Science and Art which encompasses a generous grant for the LOEWE centre Hülsdau (2007) showed that the oxidation of β-carotene “Insect Biotechnology and Bioresources”. by the purified DyP-type peroxidase MsP1 from culture supernatant can take place without addition of H O , 2 2 Publisher’s Note whereby the enzyme activity was increased by addition of Springer Nature remains neutral with regard to jurisdictional claims in pub- H O . An H O independent reaction was described for lished maps and institutional affiliations. 2 2 2 2 the oxidation of epinephrine by horseradish peroxidase Received: 27 May 2017 Accepted: 17 August 2017 or for the oxidation of Indol-3-acetic acid by plant per- oxidases (Adak et al. 1998; Gazarian et al. 1998). Here, an autocatalytic process was suggested in which superoxide radicals are formed in the presence of molecular oxygen. 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Published: Aug 23, 2017

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