Fungal strains Thermoascus aurantiacus ATCC 26904 and Thielavia terrestris NRRL 8126 were obtained from American Type Cell Culture Collection. The fungal strains were maintained on potato dextorse agar (PDA) slants at 4°C. Ethanol-washed and [C2mim][OAc]-treated switchgrass (Panicum virgatum L.) were prepared as previously described . Ammonia fiber expansion (AFEX)-treated and dilute acid-treated switchgrass were obtained as generous gifts and prepared using previously reported techniques [24, 25]. Composition (cellulose:xylan:lignin content as percentage) of each biomass substrate is as follows: untreated switchgrass (35.1:20:21.2), IL-SG (51:21.4:18.5), AFEX-SG (36:20.8:20), and dilute acid-treated switchgrass (54:7.8:31.5). All chemicals, media, and other substrates were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Thermophilic fungal cultivation
For each fungal strain, spores from a PDA slant were used to inoculate 50 mL of potato dextrose broth (PDB) and cultured at 50°C, 150 rpm. The seed culture was used to inoculate 50 mL culture flasks with biomass substrate (10 g/L), corn steep liquor (50% solids, concentrate of corn solubles) (10 g/L), sodium chloride (2 g/L), potassium phosphate (5 g/L), calcium chloride dihydrate (0.1 g/L), magnesium chloride heptahydrate (0.5 g/L), zinc (II) sulfate heptahydrate (0.002 g/L), manganese (II) chloride tetrahydrate (0.008 g/L), iron (II) sulfate heptahydrate (0.001 g/L), copper (II) sulfate heptahydrate (0.006 g/L), cobalt (II) chloride hexahydrate (0.0002 g/L) and adjusted to pH 5 prior to autoclaving. Fungi were cultured at 50°C, 150 rpm for 3 days. After cultivation, cultures were filtered with Miracloth (EMD Millipore, Billerica, MA) to remove mycelia, followed by a 0.45-μm filter with a cellulose acetate membrane (Thermo Scientific, Waltham, MA) for spore removal. The clarified supernatant was stored at 4°C. Crude enzymes were produced in duplicate cultures and used for all subsequent experiments.
Protein and glycoside hydrolase assays
Protein concentrations were determined using both Bradford assay (Bio-Rad, Hercules, CA) and bicinchoninic (BCA) assay (Pierce® BCA Protein Assay Kit, Thermo Scientific, Rockford, IL) methods using a 96-well plate (200 μL reaction volume) with bovine serum albumin as the standard. For BCA samples and standards, proteins were first precipitated with acetone and resuspended in 50 mM sodium acetate before measuring final concentration to remove interfering sugar residues . All specific activities reported in this work were calculated using Bradford protein values. Endoglucanase and xylanase activities were assessed using the DNS (3,5-dinitrosalicylic acid) method using carboxymethylcellulose and birchwood xylan as substrates, respectively, with either glucose or xylose as the standard . The enzyme reaction volume was 80 μL followed by 80 μL of DNS solution to measure released reducing sugars. One unit (U) of cellulase or xylanase activity was defined as the amount of crude protein releasing μmol of reducing sugar per min per mL of supernatant volume. Cellobiohydrolase (pNPC), β-d-glucosidase (pNPG), and β-d-xylosidase (pNPX) activities were determined using their respective p-nitrophenyl sugar substrates. 90 μL of sugar substrate was incubated with 10 μL of diluted enzyme, incubated for 30 min and quenched with 50 μL of 2% cold sodium bicarbonate. The absorbance of released p-nitrophenol was measured at 410 nm. Activities using p-nitrophenyl substrates were calculated as μmol p-nitrophenol released min-1 mg-1 crude protein. Due to possibility of color interference of the crude enzymes, samples were heat killed at 95°C overnight and used to remove background.
Saccharifications were performed in the presence of 2% (w/v) of AFEX-SG, IL-SG, or dilute-acid pretreated switchgrass. Each mixture was prepared in 50 mM sodium acetate, pH 5.0 with the designated amount of enzymes (g protein per g glucan in biomass), which were added after the reaction mixture was prewarmed to the reaction temperature to a final volume of 1 mL in a 1.5 mL Eppendorf tube. Saccharifications were carried out at specified temperatures in a shaker for 72 hours. In time-course studies on IL-SG (5 mL, performed in 15 mL Falcon tubes), 500 μL samples were removed at designated time points. All hydrolysates were collected via centrifugation at 21,000xg for 5 min and 0.45-μm filtered to remove large biomass particles prior to sugar analysis. After filtration, samples were kept frozen at −20°C and thawed prior to analysis. Glucose and cellobiose concentrations were measured on an Agilent 1200 Series HPLC system equipped with an Aminex HPX-87H column (Bio-Rad) and Refractive Index Detector. Samples were run with an isocratic 4 mM sulfuric acid mobile phase. Sugar concentrations were determined using standards containing both glucose and cellobiose. Glucan conversion (mass of glucose plus cellobiose) was calculated using estimated glucan contents of pretreated switchgrass.
Proteomic analysis of thermophilic fungal secretomes
For SDS-PAGE analysis, supernatants were concentrated 50-fold with 10,000 MWCO PES ultrafiltration column (Vivaspin 500, Sartorius, Germany), loaded onto an 8 – 16% Tris-glycine Mini gel (Invitrogen, Carlsbad, CA) and run at 130 V for 90 minutes. Gels were stained with GelCode Blue Safe Protein Stain (Thermo Scientific). After staining and extensive rinsing, target protein bands were excised and digested using trypsin .
Samples were analyzed on an ABSciex TripleTOF 5600 (ABSciex, Foster City, CA) coupled to Eskigent nano-lc systems (Applied Biosystems, Foster City, CA). Peptide samples were injected onto a Pepmap100 μ-guard column (Dionex, Sunnyvale, CA) via a Famos Autosampler (Dionex) and washed for 10 minutes with Buffer A (2% Acetonitrile, 0.1% Formic Acid) at 15 μL/min. Peptides were eluted onto an Acclaim Pepmap100 C18 column (75 μm x 150 mm, 300 nL/min flow rate; Dionex) via a gradient consisting of initial starting condition of 5% buffer B (98% Acetonitrile, 0.1% Formic Acid), increasing B to 10% in 2 minutes followed by a 58 minute ramp to 35% B. Subsequently, B was increased to 90% over 3 minutes and held for 7 minutes followed by a ramp back down to 5% B for 15 minutes to re-equilibrate the column to the original condition. Peptides were introduced to the mass spectrometer by using a Nanospray III source (ABSciex) operating in positive-ion mode (2400 V) and data acquired with Analyst TF 1.5.1. The TripleTOF 5600 was operated in information dependent acquisition (IDA) mode whereby the ten most intense ions within 400 m/z to 1250 m/z mass range exceeding 150 counts (charge states 2–5) were selected for MS/MS analysis (high sensitivity mode, UNIT resolution with rolling collision energy). MS/MS spectra were scanned from 100 m/z to 1600 m/z and were collected for a total accumulation time of 200 ms. Former parent ions were excluded for 16 seconds following MS/MS acquisition. The acquired spectra were exported as.mgf files from Peakview version 1.1.1 (ABSciex) and processed with Mascot version 2.3.02 with a peptide tolerance of ±50 ppm and MS/MS tolerance of ±0.1 Da; variable modification was Oxidation (M) with up to one missed cleavage for trypsin. As a limited number of protein sequences from Thermoascus aurantiacus are available, searches were performed against sequenced T. aurantaicus proteins available in GenBank combined with a full ORF database from Thielavia terrestris obtained through the Genome Portal of the Joint Genome Institute , a list of common contaminants (e.g., trypsin, human keratin) and consisted of 10,007 sequences. Mascot DAT files were uploaded into Scaffold version 3.5 and searched via X!Tandem with the same sequences and parameters.