A Fusarium graminearum-based enzyme blend for the efficient hydrolysis of hemicellulose, a crucial step for competitive bioethanol production, is described. The heretofore-uncharacteri zed endo-1,4-beta-xylanase (XylD), 1,4-beta-xylosidase (XyloA), and bifunctional xylosidase/arabinofuranosidase (Xylo/ArabA) were produced at high levels in Escherichia coli (10-38 mg/l). They displayed compatible pH and temperature-dependences, allowing their utilization for simultaneous substrate digestions. Monosaccharide analysis indicated a strong positive synergism between the enzymes during the degradation of oat spelt xylan. Two units of each protein catalyzed the release of 61% and 15% of the total amount of available D-Xylose and L-arabinose, respectively, in only 4 h. The detailed cooperative mechanism of the three hydrolases was elucidated by polysaccharide analysis using carbohydrate gel electrophoresis (PACE) and the enzymes were shown to be suitable for the partial hydrolysis of pretreated crude plant biomass. (C) 2008 Elsevier Ltd. All rights reserved.