reesei, were shown to be responsible for postsecretorial modifications of glycan structures. In the present study, a glycoside hydrolase family 18 ENGase (accession number CAZ16624) was partially purified from the culture medium of T. reesei Rut-C30. The enzyme was denoted Endo T for endoglycosidase of T. reesei. The gene can be found in the T. reesei genome on scaffold 15, where the protein (ID 44979) is annotated as distantly related to chitinases (Martinez et al., 2008). The purification and characterization of this fungal ENGase as well as the relationship with other family 18 members are described. To study the distribution of deglycosylating Opaganib purchase activity within filamentous fungi, spores of three

cellulolytic organisms (Fusarium oxysporum MUCL 14162, Humicola insolens MUCL 8343 and Phanaerochaete chrysosporium MUCL 19343), one fungus with reported ENGase activity (Aspergillus oryzae MUCL 31310) and four species with a homologous sequence as identified by blast search [Neurospora crassa MUCL 19026, Magnaporthe

grisea GUY II (Leung et al., 1988), Gibberella zeae and Aspergillus nidulans MUCL 20209] were inoculated and grown directly into Sabouraud-dextrose broth (Oxoid). Also, eight Trichoderma and Hypocrea species LEE011 belonging to the three phylogenetic sections and to different clades (Trichoderma pseudokoningii CBS 408.91, Trichoderma longibrachiatum CBS 816.68, T. reesei CBS 383.78, Trichoderma atroviride P. Karst 1892 CBS 142.95, Trichoderma koningii CBS 457.96, Trichoderma hamatum CBS 102160 (= DAOM 167057), Trichoderma harzianum CBS 226.95 and Trichoderma crassum CBS 336.93) were cultivated in Sabouraud medium. These vouchered Trichoderma and Hypocrea strains are described and identified by DNA analyses [e.g. 18S rRNA gene and internal transcribed spacer (ITS)1 and ITS2 DNA] in references Lieckfeldt et al. (1992), Kuhls pheromone et al. (1996), Kindermann

et al. (1998), Kullnig-Gradinger et al. (2002) and Druzhinina et al. (2005) and reflect the biodiversity in Trichoderma and Hypocrea species (Druzhinina et al., 2005). Culture filtrate from a fed-batch fermentation of T. reesei Rut-C30 was set up by Iogen Corporation (Ottawa, ON, Canada) as described before (Hui et al., 2001). A sample was harvested 44 h after induction of cellulase production. Three hundred milliliters of extracellular medium (15 mg protein mL−1), 100 g Avicel and 100 mL 100 mM sodium acetate, pH 5, were incubated overnight at 4 °C. The nonbound fraction was separated by centrifugation (S1). The Avicel with the bound proteins was washed with buffer for 1 h at 4 °C and the supernatants were collected by centrifugation (S2). The combined nonbound fractions (S1+S2) were loaded on a DEAE-Sepharose FF (10 × 1 cm) column equilibrated with 5 mM ammonium acetate and subsequently eluted with a linear gradient of 5–300 mM ammonium acetate, pH 5 (flow rate 1.5 mL min−1). The concentrated active fractions were applied to a Biogel P-100 column (75 × 0.75 cm) and eluted at 0.