The synergistic associations between two hemicellulases and endoglucanase E from Clostridium cellulovorans on various substrates were investigated. The recombinant enzymes were expressed, purified using affinity chromatography and tested for activity against sugarcane bagasse, xylan, carboxymethycellulose and locust bean gum. The results obtained illustrated synergistic associations between the three recombinant cellulosomal enzymes. Novel synergistic associations were established between mannanase A and encloglucanase E, as well as between xylanase A and mannanase A, in the degradation of sugarcane bagasse, xylan, carboxymethylcellulose and locust bean gum. The combination with the molar ratio of xylanase A to endoglucanase E of 75%:25% against sugarcane bagasse produced the greatest synergistic association (degree of synergy of 3.59). However, a molar ratio of xylanase A to endoglucanase E to mannanase A of 25%:25%:50% produced the largest degree of synergy against sugarcane bagasse and the mixed substrate containing 2% (w/v) birchwood xylan, 2% (w/v) carboxymethylcellulose and 2% (w/v) locust bean gum. These optimal enzyme combinations are important for the optimal release of reducing sugars from lignocellulosic waste materials such as sugarcane bagasse for subsequent biofuel production. (c) 2008 Elsevier Inc. All rights reserved.