Signal peptide predication and activity assays.

Signal peptides were predicted using LipoP, version 1.0 (http://www.cbs.dtu.dk/services/LipoP/) (76) Enzymatic assays were performed using 4-nitrophenyl derivatives as substrates (Sigma-Aldrich, Saint Louis, MO, USA). Initially, 50 μl of partially purified protein solution was incubated with 100 μl of citrate phosphate buffer, pH 7 (0.1 M citric acid, 0.2 M dibasic sodium phosphate), and 50 μl of substrate (Table S3). A microplate was incubated at 37°C in a Tecan Spark 20 M microplate reader (Tecan, Mannedorf, Switzerland). Th rate of released 4-nitrophenol was monitored at 405 nm. Assays were performed in triplicate.

Following previous studies (77, 78), enzymatic activity was also assessed by the release of reducing sugars. CMC (Sigma-Aldrich, Saint Louis, USA), arabinoxylan, galactomannan, glucomannan, and xylan (Megazyme, Wicklow, Ireland) were used as substrates. Briefly, 100 μl of partially purified enzyme solution was incubated with 50 μl of citrate phosphate buffer, pH 7 (0.1 M citric acid, 0.2 M dibasic sodium phosphate), and 100 μl of substrate at 37°C for 30 min (Table S3). The concentrations of reducing sugars were determined applying the Somogyi-Nelson method, and absorbance was read at 620 nm using Specord Plus spectrophotometer (Analytik Jena, Jena, Germany). Assays were performed in triplicates.

Single enzymes as well as enzymatic cocktails were used for the pretreatment of glucomannan to determine the release of d-glucose, d-mannose, and acetic acid. Briefly, pretreatment was carried out in 500 μl as follows: 250 μl of substrate, 50 μl of buffer, and 50 μl of each enzyme assessed; final volume was reached by addition of water if necessary. The reaction mixture was incubated at 37°C for 1 h. If a cascade reaction was tested, a second enzyme (enzymatic set) was added after 1 h of pretreatment and reincubated for 1 h at 37°C. The release of d-glucose, d-mannose, and acetic acid was determined using commercially available kits (Megazyme, Wicklow, Ireland), according to the supplier’s instructions. An enzymatic cocktail containing uBac-GH3, uBac-CE6, uBac-GH26a, and uBac-GH26b was taken as a reference to normalize results to 100%.