Enzyme expression, production and purification
MtLPMO9A from Myceliophthora thermomphila C1 (UniProt: KP901251) was over-expressed in a protease/(hemi-) cellulase-free C1-expression host (LC strain) [40, 41]. The C1 strain was grown aerobically in 2-L fermentors using a medium containing glucose and ammonium sulfate, and enriched with essential salts [41]. Enzyme production was performed under glucose limitation in a fed-batch process (pH 6.0; 32°C) as described previously [40] and resulted in an MtLPMO9A-rich crude enzyme extract. The crude enzyme extract was dialyzed against 10 mM potassium phosphate buffer (pH 7.0). MtLPMO9A was purified using an AKTA-Explorer preparative chromatography system (GE Healthcare, Uppsala, Sweden). As a first step, 3 g of the dialyzed crude enzyme mixture (50 mg mL−1) was subjected to a self-packed Source 15Q column (100 × 70 mm internal diameter, GE Healthcare), pre-equilibrated in 20 mM potassium phosphate buffer (pH 7.0). After protein application, the column was washed with three column volumes of 20 mM potassium phosphate buffer (pH 7.0). Elution was performed with a linear gradient of 0–1 M NaCl in 20 mM potassium phosphate buffer (pH 7.0) over five column volumes at 25 mL min−1. The eluate was monitored at 220 and 280 nm. Fractions (20 mL) were collected and immediately stored on ice. Peak fractions were pooled and concentrated using ultrafiltration (Amicon Ultra, molecular mass cut-off of 3 kDa, Merck Millipore, Cork, Ireland) at 4°C. The concentrated pools were subjected to SDS-PAGE (Additional file 1). For further purification (2nd step), the MtLPMO9A-containing pool (fraction AEC-I, Additional file 1) was loaded onto a self-packed Superdex TM-75 column (100 × 3 cm internal diameter, GE Healthcare) and eluted at 5 mL min−1 with a 10 mM potassium phosphate buffer (pH 7.0) containing 150 mM NaCl. Fractions (5 mL) were immediately stored on ice. Peak fractions were pooled and concentrated by ultrafiltration as described above.
The MtLPMO9A preparation thus obtained (partially purified fraction SEC-I; Additional file 1) was further subjected (3rd step) to a Resource Q column (30 × 16 mm internal diameter, GE Healthcare), pre-equilibrated in 20 mM potassium phosphate buffer (pH 7.0). After protein application, the column was washed with 20 column volumes of starting buffer. Elution at 6 mL min−1 was performed with a linear gradient of 0–1 M NaCl in 20 mM potassium phosphate buffer (pH 7.0) over 20 column volumes. Elution was monitored at 220 and 280 nm. Fractions (3 mL) were immediately stored on ice. Peak fractions were pooled and concentrated by ultrafiltration as described above.
Protein identification
Sequencing of the MtLPMO9A coding sequence was carried out by the Scripps Research Institute, USA.
Protein content
To analyze protein contents, the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) was used with bovine serum albumin (BSA) as calibration.
SDS-PAGE
The protein purity was analyzed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore, proteins were reduced with β-mercaptoethanol, heated for 10 min and loaded on 12% polyacrylamide gels (Mini-PROTEAN TGX Gels, Bio-Rad Laboratories, Hempel Hempstead, UK). In addition, a protein marker (Protein All Blue Standards, Bio-Rad Laboratories) was loaded for mass calibration. Gels were stained with the EZBlue Gel Staining Reagent (Sigma Aldrich, Steinheim, Germany).
LC/ESI–MS
Purified MtLPMO9A (2.5 mg mL−1 in 0.1% (v/v) trifluoroacetic acid (TFA) in H2O) was analyzed by liquid chromatography/electron spray ionization-mass spectrometry (LC/ESI–MS) using an ACQUITY UPLC separation system (Waters, Milford, MA, USA) equipped with a C4-reversed phase column (UPLC BEH C4 1.7μm, 2.1 × 100 mm, Waters) coupled to a PLC LG 500 photodiode array detector (Waters) and a SYNAPT G2-Si High Definition Mass Spectrometer (Waters). Gradient elution with eluent A (H2O + 1% (v/v) acetonitrile + 0.1% (v/v) TFA) and eluent B (acetonitrile + 0.1% (v/v) TFA) was performed according to the following steps: From 0 to 2 min isocratic 90% A, from 2 to 12 min gradient from 90% A to 25% A, from 12 to 15 min gradient from 25% A to 100% B and from 12 to 15 min isocratic at 100% B; then re-equilibration to the initial conditions. The flow rate and the injection volume were 0.35 mL min−1 and 2 μL, respectively. The photodiode array detector was operated at a sampling rate of 40 points sec−1 in the range 200–400 nm, resolution 1.2 nm. The SYNAPT mass spectrometer was operated in the positive ion mode (resolution mode), capillary voltage 3 kV, sampling cone 30 V, source temperature 150°C, desolvation temperature 500°C, cone gas flow (N2) 200 L h−1, desolvation gas flow (N2) 800 L h−1, acquisition in the full scan mode, scan time 0.3 s, interscan time 0.015 s, acquisition range 150–4,000 m/z.
Substrates incubated with MtLPMO9A
OSX, BiWX, Avicel PH-101, xylo-oligosaccharides (DP1-5) and β-(1 → 4)-linked gluco-oligosaccharides (DP1-5) were obtained from Sigma-Aldrich (Steinheim, Germany). WAX (medium viscosity), β-(1 → 3, 1 → 4)-linked glucan from barley (medium viscosity) and oat spelt (medium viscosity) were purchased from Megazyme (Bray, Ireland). Xyloglucan (XG; from tamarind seed) was obtained from Dainippon Sumitomo Pharma (Osaka, Japan). Regenerated amorphous cellulose (RAC) was prepared from Avicel PH-101 by adopting a method described elsewhere [42]. Briefly, Avicel PH-101 (100 mg) was moistened with 0.6 mL water. Next, 10 mL 86.2% (w/v) ortho-phosphoric acid was slowly added followed by rigorous stirring for 30 min until the cellulose was completely dissolved. The dissolved cellulose precipitated during stepwise addition of 40 mL of water. After centrifugation (4,000g, 12 min, 4°C), the pellet obtained was washed twice with water and neutralized (pH 7.0) with 2 M sodium carbonate. The pellet was washed again with water (three times) and the final pellet was suspended in water to a dry matter content of 1.4 ± 0.1% (w/w) RAC suspension.
MtLPMO9A activity assays
Substrates (1–2 mg mL−1, see Figure captions) were dissolved in 50 mM ammonium acetate buffer (pH 5.0), with or without addition of ascorbic acid (final concentration of 1 mM). MtLPMO9A was added (12.5 µg mg−1 substrate) and incubated for 24 h at 50°C in a head-over-tail rotator in portions of 1 mL total volume (Stuart rotator, Bibby Scientific, Stone, UK) at 20 rpm. Supernatants of all incubations, including substrates incubated with and without ascorbic acid in the absence of MtLPMO9A, were analyzed by HPAEC and MALDI-TOF MS.
Structural modelling
An alignment was made of the amino acid sequence of MtLPMO9A and the amino acid sequence of PMO1 from Thielavia terrestris, which scored highest in a Blast search using the MtLPMO9A sequence against the Protein Data Bank (75% amino acid identity). Using this alignment and the available structure of TtPMO1 (PDB-id: 3eii) as template, structural models were obtained for MtLPMO9A using the Modeller program version 9.14 [43]. Thirty comparative models were generated, after which the model with the lowest corresponding DOPE score [44] was selected for image generation using Pymol (Pymol, The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC, New York, NY, USA).
Oligosaccharides analysis
Oligosaccharides were analyzed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The HPAEC system (ICS-5000, Dionex, Sunnyvale, CA, USA) was equipped with a combination of a CarboPac PA1 guard column (50 mm × 2 mm i.d., Dionex) and a CarboPac PA1 analytical column (250 mm × 2 mm i.d., Dionex). The flow rate was 0.3 mL min−1 (20°C). Samples were kept at 6°C in the autosampler and the injection volume was 10 µL. Elution was performed using two mobile phases: 0.1 M NaOH and 1 M NaOAc in 0.1 M NaOH. The gradient elution program was as follows: 0–30 min, linear gradient 0–400 mM NaOAc; 30–40 min linear gradient 400–1,000 mM NaOAc; followed by a cleaning step and equilibration (15 min) of the column with the starting conditions. Soluble gluco- and xylo-oligosaccharides (degree of polymerization 1–5) as well as glucuronic and gluconic acid were used as standards (Sigma-Aldrich).
MALDI-TOF MS
For matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), an Ultraflex workstation using FlexControl 3.3 (Bruker Daltonics) equipped with a nitrogen laser of 337 nm was used. The pulsed ion extraction was set on 80 ns. Ions were accelerated to a kinetic energy of 25 kV and detected in positive reflector mode with a set reflector voltage of 26 kV. The lowest laser energy required was used to obtain a good signal-to-noise ratio. A total of 200 spectra were collected for each measurement. The mass spectrometer was calibrated using a mixture of maltodextrins (Avebe, Veendam, The Netherlands) in a mass range (m/z) of 500–2,500. The peak spectra were processed by using FlexAnalysis software version 3.3 (Bruker Daltonics). Prior to analysis, samples were desalted by adding AG 50 W-X8 Resin (Bio-Rad Laboratories). To obtain lithium (Li) adducts, the supernatant was dried under nitrogen and re-suspended in 20 mM LiCl [28]. Each lithium-enriched sample of a volume of 1 µL was mixed with 1 µL of matrix solution (12 mg mL−1 2,5-dihydroxy-benzoic acid (Bruker Daltonics) in 30% (v/v) acetonitrile in H2O), applied on an MTP 384 massive target plate (Bruker Daltonics) and dried under a stream of warm air.