0, were incubated for 10 min at 30 °C. The reaction was stopped by addition of 250 μL 1.0 M NaOH and incubation was continued at 96 °C for 5 min and A405 nm of the reaction mixture then measured. One unit of

xylanase was defined as the amount of enzyme required to release 1 μmol reducing sugar min−1 under the assay conditions; xylose was used as a standard (ɛ405=2.81 mM−1 cm−1). Glucoamylase activity was measured as described previously (Yoon et al., 2006). Culture filtrates (20 μL) and 0.1% w/v amylose (Mw=c. 2800, Tokyo Chemical Industry Co. Ltd, Tokyo, Japan) in 100 mM sodium acetate, pH 5.0, were incubated for 30 min at 30 °C. After incubation, the concentration of glucose was estimated with a Glucose CII-Test Wako (Wako Pure Chemical Industries Ltd) based on the glucose oxidase method. One unit of glucoamylase was defined as the amount of enzyme required Sirolimus to release 1 μmol glucose min−1 under the assay conditions. Culture filtrates from

medium containing cellulose (C), cellulose+xylan (CX) and cellulose+starch (CS) were centrifuged at 15 000 g for 5 min at 4 °C to remove insoluble materials. The supernatants were then concentrated using Selleckchem Trichostatin A a 10 kDa Ultrafree®-0.5 Centrifugal Filter Device (Millipore, Billerica, MA) and washed with Milli-Q water three times. Samples were examined on a Multiphor system (GE Healthcare UK Ltd, Buckinghamshire, UK). Proteins (25 μg) were mixed with a rehydration buffer containing 7.5 M urea, 2 M thiourea, 4% CHAPS, 2% dithiothreitol, 0.5% IPG buffer (GE Healthcare UK Ltd) and a trace amount of bromophenol blue to a final volume of 330 μL and then loaded onto Immobiline Drystrips (18 cm, pH 3–10, nonlinear; GE Healthcare UK Ltd). After rehydration for 12 h, proteins were isoelectrically focused under the following conditions: 500 V (gradient over 1 min); 3500 V (gradient over 90 min); 3500 V (fixed for 6 h). These strips were equilibrated with buffer I [50 mM Tris–HCl pH 6.8, 6 M urea, 2% w/v sodium dodecyl sulfate (SDS), 30% w/v glycerol, 2% w/v dithiothreitol] and then Janus kinase (JAK) buffer II (50 mM Tris–HCl pH 6.8, 6 M urea, 2% w/v SDS, 30% w/v glycerol, 2.5% w/v iodoacetamide). These strips were

placed on SDS-polyacrylamide gels (ExcelGel™ SDS XL 12-14; GE Healthcare UK Ltd) and electrophoresis was conducted under the following conditions: 12 mA for 60 min, 40 mA for 5 min and finally 50 mA for 160 min. The gels were fixed in 10% v/v acetic acid and 40% v/v EtOH and then stained with SYPRO Ruby (Bio-Rad Laboratories) for 1 h. The staining solution was removed, and the gels were washed in 10% acetic acid and 10% v/v MeOH solution for 30 min. The stained 2DE gels were scanned with excitation at 532 nm using a Typhoon image scanner (GE Healthcare UK Ltd) and individual protein spots on different gels were matched and quantified using progenesis samespots ver. 4.0 (Nonlinear Dynamics Limited, Durham, NC). The protein spots were excised, washed in 200 μL acetonitrile and then dried under vacuum.