Generation of a fermentable hydrolysate from arabinoxylan is a first prerequisite in the utilization of wheat hemicellulose in the biofuel industry. This study examined the efficacy of four hemicellulolytic microbial enzyme preparations and one xylanase preparation in catalyzing degradation of purified water soluble and water insoluble wheat arabinoxylan-with particular emphasis on the catalytic degradation of water insoluble arabinoxylan. The effects of individual enzyme treatments were compared by assessing yields of arabinose, xylose, and xylobiose obtained under different reaction conditions of pH and temperature in response surface designs. In general, the monosaccharide yields obtained were lower on the water insoluble wheat arabinoxylan than on the water soluble. On both substrates, the Ultraflo L preparation from Humicola insolens was best in catalyzing arabinose and xylobiose release, while the Celluclast 1.5 L preparation from Trichoderma reesei was superior to all the other enzyme preparations in catalyzing xylose release. Treatments with 50:50 combinations of the enzyme preparations only resulted in a pronounced synergistic xylose release with a mixture of Ultraflo L:Celluclast 1.5 L. Examination of the progress of the substrate degradation indicated that the synergism on the insoluble arabinoxylan resembled what we have previously observed on soluble arabinoxylan, i.e. that the effect was a result of positive interaction in arabinose release and xylan depolymerization between the alpha-L-arabinofuranosidase (EC 3.2.1.55) and endo-1,4-beta-xylanase (EC 3.2.1.8) activities present in Ultraflo L, and between the beta-xylosidase (EC 3.2.1.37) activity present in the Celluclast 1.5 L. The results of treatments with combinations of Ultraflo L and purified T reesei beta-xylosidase - with both simultaneous and sequential addition, and with and without contemporary pH adjustments to optimize individual enzyme activities - strongly corroborated this conclusion. (c) 2006 Elsevier Inc. All rights reserved.