Organism
Marasmius rotula [Scop.] Fr. (Pinwheel mushroom) (deposited at the German collection of microorganisms and cell cultures--DSMZ, collection number DSM 25031) was isolated from fruiting bodies that had developed on a meadow near Senftenberg (Germany) containing subsurface woody debris of Rubinia pseudoacacia (False Acacia). To confirm the strain affiliation to M. rotula at molecular level, the complete internal transcribed spacer (ITS) region including the 5.8S rRNA sequence of the ribosomal DNA was analyzed. For this, the isolated culture was grown on agar containing 1% malt extract and 0.5% glucose. Thereafter, genomic DNA was extracted from 100 mg homogenized fungal mycelium using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. PCR amplification of genomic DNA was performed in a total volume of 20 μl containing 2 μl template, 9 μl GoTaq Green Master Mix, and 0.4 μl 25 pmol of each of two general fungal ITS-rDNA primers ITS1 and ITS4 (White et al. 1990) using standard cycling conditions. The direct sequencing of resulting 777 bp amplicon cleaned with ExoSAP-IT (USB Europe GmbH, Staufen, Germany) was performed on an ABI PRISM 3730 × l Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems). The quality of the obtained sequence was checked by visual inspection of the electropherogram using a Sequence Scanner v.1.0 (Applied Biosystems) and edited using BioEdit v.7.0.9 (Hall 1999). A nucleotide BLAST search at GenBank database (Zhang et al. 2000) in September 2011 revealed several hundred homologous sequences from Marasmius and related genera. However, only two sequences in the database reply showed a high query coverage (representing more than 700 aligned nucleotides) and a sequence identity higher than 95%: an unnamed Marasmius species (accession number DQ449983, 709 aligned nucleotides, 99% sequence identity) and the only known ITS sequence in GenBank of M. rotula (accession number DQ182506, 701 aligned nucleotides, 98% sequence identity) provided by the AFTOL project (Assembling the fungal tree of life; Celio et al. 2006). The ITS sequence of M. rotula strain DSM 25031 was submitted to GenBank database (accession number JN714927).
Culture conditions
Fungal stock cultures were grown in culture slants on 2% malt extract agar at 24°C and stored at 4°C in the dark. The content of one slant was homogenized in 40 ml sterile sodium chloride solution (0.9%) and used for the inoculation of liquid cultures (5% vol/vol of the mycelium suspension). The carbon- and nitrogen-rich basic liquid medium contained 28 g L-1 glucose; 12 g L-1 peptone from soybean (Roth, Karlsruhe, Germany) and 3 g L-1 yeast extract (Merck, Darmstadt, Germany) dissolved in distilled water (Ikehata et al. 2004). Enzyme production was performed in 500-mL Erlenmeyer flasks containing 200 mL of the liquid medium. Cultivation occurred on a rotary shaker (120 rpm) at 24°C for three to four weeks. Peroxygenase activity was measured every 1 to 3 days.
M. rotula was also cultivated in 5-L stirred-tank bioreactors (Sartorius, Melsungen, Germany) to produce larger amounts of peroxygenase. The medium (4 L) was the same as above and inoculation occurred with 200 ml mycelial suspension (fermentation parameters: 24°C, stirring at 300 rpm and 100% dissolved oxygen without pH-regulation).
Enzyme assays
Peroxygenase activity was routinely measured by following the oxidation of veratryl alcohol into veratraldehyde (ε310: 9,300 M-1 cm-1) in McIlvaine buffer at pH 5.5 (Ullrich et al. 2004). Reaction was started by addition of 2 mM H2O2. Laccase activity in the culture liquid and during the purification was detected by the oxidation of ABTS in McIlvaine buffer at pH 4.5 in the absence of H2O2 (Majcherczyk et al. 1999).
Further activities of purified M. rotula aromatic peroxygenase (Mro APO) were measured with benzyl alcohol, 2,6-dimethoxyphenol (DMP) and ABTS under identical conditions by monitoring the formation of benzaldehyde (ε280: 1,400 cm-1 M-1), dimeric DMP quinone (ε469: 49,600 M-1 cm-1) and the ABTS cation radical (ε420: 36,000 M-1 cm-1) (Ullrich et al. 2004). Ring-hydroxylating activity of Mro APO was determined by the peroxygenation of naphthalene to 1-naphthol (ε303: 2,030 M-1 cm-1). The assay mixture contained in a total of 2 ml: 1 ml sodium phosphate/citrate buffer (100 mM pH 5.5), 200 μl naphthalene (5 mM) dissolved in 100% acetonitrile and 10 to 100 μl enzyme solution. The reaction was started with 20 μl H2O2 (200 μM) (Kluge et al. 2007).
Enzyme purification
The culture liquid of M. rotula was separated from the mycelium by filtration through paper filters (GF8, Whatman GmbH, Dassel, Germany). The filtrate was frozen at -80°C and then re-thawed to precipitate and remove extracellular glucans. After thawing, the culture liquid was filtrated through glass fiber filters (GF 8, Whatman).
The filtrate was concentrated 40-fold by two steps of ultrafiltration using two tangential-flow cassettes (Sartocon Slice Cassette, Hydrosart, cut-off 10 kDa, Sartorius, and Omega membrane, cut-off 10 kDa, Pall Life Sciences, Dreieich, Germany). All subsequent chromatographic purification steps were performed with an Äkta FPLC™System (GE Healthcare Europe GmbH, Freiburg, Germany). In the first step, the crude preparation was loaded onto a Q Sepharose FF column (anion exchanger XK 26/20, GE Healthcare) and the proteins were eluted with a linear gradient of 0-0.7 M NaCl in 10 mM sodium acetate buffer (pH 5.5) at a flow rate of 6 ml min-1. Mro APO containing fractions were pooled, concentrated and dialyzed against 10 mM sodium acetate (pH 5.5, 10 kDa cut-off Omega membrane, Pall Life Sciences). Pooled fractions were subjected to a second anion exchanger consisting of mono beads (Mono Q 10/100, GE Healthcare) and using sodium acetate (10 mM, pH 5.5) and an increasing gradient up to 0.4 M sodium chloride for elution. Mro APO containing fractions were pooled, concentrated and dialyzed against 10 mM sodium acetate. In the next step, an anion exchanger consisting of Source 15Q (10/10, GE Healthcare) was used along with sodium acetate (25 mM, pH 5.0) and an increasing gradient up to 0.4 M sodium sulphate for elution. Mro APO containing fractions were pooled, concentrated and dialyzed against 50 mM sodium phosphate buffer (pH 8.0, 10 kDa cut-off Vivaspin 20, Satorius Stedim Biotech GmbH, Goettingen, Germany). Afterwards, size exclusion chromatography (SEC; Superdex 75 16/60, GE Healthcare) was carried out under isocratic conditions in 50 mM sodium phosphate (pH 8.0, +0.15 M sodium chloride). The pooled and concentrated Mro APO fractions were re-chromatographed on a Mono Q column (5/50, GE Healthcare) using sodium acetate (10 mM, pH 5.5) and an increasing gradient up to 0.25 M sodium chloride for elution. Mro APO containing fractions were eventually pooled, concentrated and dialyzed against 50 mM sodium phosphate buffer (pH 8.0, 10 kDa cut-off Vivaspin 20) and stored at 4°C. During early FPLC-separation steps (e.g. first Mono-Q separation), the elution profiles showed up to four heme peaks with veratryl alcohol oxidizing activity; we focused our purification efforts always on the most active fraction (i.e. that with the highest specific activity).
Characterization of Mro APO
Molecular mass of purified Mro APO was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12.5% NuPage Novex Bis-Tris Gel (Invitrogen, Darmstadt, Germany). A low-molecular mass protein calibration kit (MBI Fermentas, PageRuler™, Roth, Germany) was used as standard. Isoelectric focusing (IEF) was carried out using precast gels (pH 3-7; Invitrogen) and specific IEF markers (pH 3-10; Serva, Heidelberg, Germany) as standard. Electrophoretically separated protein bands were visualized with the Colloidal Blue staining kit (Invitrogen).
Purified Mro APO (75 μg) was denaturated with SDS and deglycosylated for 4 hours with the Enzymatic Protein Deglycosylation Kit from Sigma (Saint Louis, MO, USA). The latter contained PNGase F, O-glycosidase, two α-2(3,6,8,9) neuramidases as well as β-1,4-galactosidase and β-N-acetylglucosaminidase and was used according to the instructions of the provider. Molecular mass of the deglycosylated protein was determined by SDS-PAGE using a 12% NuPAGE Novex Bis-Tris Gel (Invitrogen).
Apparent Michaelis-Menten (Km) and catalytic (kcat) constants of purified Mro APO were determined at pH 5.5 for veratryl alcohol, benzyl alcohol, DMP, ABTS, naphthalene, and H2O2. Lineweaver-Burk plots were prepared from the initial rates obtained with various substrate concentrations while the concentration of the second substrate was kept constant.
Stability of Mro APO in organic solvents was tested by incubating the enzyme (1 U = 0.31 μM) in aqueous buffer mixtures (vol/vol) of methanol, acetonitrile and N,N-dimethylforamide (DMF). Organic solvent concetrations of 10, 30, 50 and 70% were used. All mixtures were kept at 24°C for up to 120 min. For determination of the remaining enzyme activity, samples were taken after 1, 30, 60 and 120 min and measured using the veratryl alcohol assay mentioned above.
The UV-Vis spectrum of the resting-state Mro APO was recorded in 10 mM sodium acetate buffer (pH 5.5) in the range from 200 to 700 nm using a Cary 50 spectrophotometer (Varian, Darmstadt, Germany). To obtain the reduced CO-enzyme complex, samples were reduced with sodium dithionite and flushed with carbon monoxide for 2 min.
For N-terminal analysis, Mro APO was separated by SDS-PAGE as described above and transferred from the SDS gel to a polyvinylidene fluoride membrane (Amersham Biosciences, Freiburg, Germany) by electroblotting. Sequencing by Edman degradation and de-novo-peptide sequencing using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF/TOF) after digestion with trypsin were performed by Protagen AG (Dortmund, Germany). Sequences were compared with known APOs (A. aegerita, C. radians) and chloroperoxidase (CPO; Caldariomyces fumago) as well as with putative APO- and CPO-like sequences from the National Center for Biotechnology Information (NCBI) GenBank database by using BLASTp.
Further substrate oxidation studies
Enzymatic conversion of toluene was performed in 2-mL reaction vials containing citrate phosphate buffer (pH 5.5), 1 mM toluene, and 10 U Mro APO (3.1 μM). The reaction was started by addition of H2O2 (2 mM), which was repeated four times in 2.5 min steps.
For naphthalene oxidation, two reaction solutions were prepared. Solution A contained 2 U Mro APO (0.62 μM) in 1 mL citrate phosphate buffer (pH 5.5) and solution B consisted of 2 mM naphthalene and 2 mM H2O2 in 1 mL 50% (vol/vol) acetonitrile. Every 2.5 min, 250 μL of solution B were transferred to solution A. After 10 min, reactions were stopped by addition of 20 μl HCl (37%).
Reaction products were analyzed by HPLC using an Agilent® 1200 system (Waldbronn, Germany) equipped with a diode array detector and a LiChrospher reversed phase (C18) column (4.6 × 125 mm, 5 μm; Phenomenex, Aschaffenburg, Germany). For toluene and its oxidation products, a gradient separation was applied from 15 to 80% acetonitrile (0-5 min; 15%; 5-25 min 15-80%) in 20 mM aqueous potassium dihydrogenphosphate buffer (pH 2.8) at a flow rate of 0.7 mL min-1. For naphthalene, a gradient from 20 to 80% acetonitrile (0-5 min; 20%; 5-20 min; 20-80%) in 20 mM potassium dihydrogen phosphate (pH 2.8) was used at a flow rate of 1 mL min-1. Eluted substances were recorded at 220 nm and identified/quantified by means of authentic standards.
Possible halogenating activity of Mro APO was tested as previously described (Ullrich and Hofrichter 2005). Briefly, the enzyme (0.31 μM) was incubated in potassium phosphate buffer (100 mM, pH 3) in the presence of phenol (0.1 mM), potassium bromide or chloride (10 mM) and H2O2. After 10 min, the reaction mixture was analyzed by HPLC for the formation of bromo- and chlorophenols using authentic monohalophenols as standards. In a second test, the oxidation of bromide (Br-) and iodide (I-) into tribromide (Br3
-) and respectively triodide (I3
-) was spectrophotometrically followed (Libby et al. 1982).
Chemicals
2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was purchased from Applichem (Darmstadt, Germany), 2,6-dimethoxyphenol (DMP), naphthalene, 1-napththol, 2-naphthol, potassium chloride and toluene from Sigma-Aldich (München, Germany). Veratryl alcohol, veratraldehyde, veratric acid, benzyl alcohol, benzaldehyde, benzoic acid, phenol, H2O2 (30%, w/v), sodium acetate, sodium chloride, sodium sulphate, methanol and acetonitrile were obtained from Roth (Steinheim, Germany). All other chemicals and solvents were purchased from Merck (Darmstadt, Germany).