Cassava pulp, a solid by-product from starch processing, is a promising and underused biomass that can be converted to biofuels and other value-added bio-products. In this study, an alternative cassava pulp saccharification process, which utilizes the multi-activity enzyme from Aspergillus niger BCC17849 and obviates the need for a pre-gelatinization step, was developed. The crude multi-enzyme composed of non-starch polysaccharide hydrolyzing enzyme activities, including cellulase, pectinase and hemicellulase act cooperatively to release the trapped starch granules from the fibrous cell wall structure for subsequent saccharification by raw starch degrading activity. A high yield of fermentable sugars, equivalent to 716 mg glucose and 67 mg xylose/g of cassava pulp, was obtained after 48 h incubation at 40 degrees C and pH 5 using the multi-enzyme, which was greater than the yield obtained from the optimized combinations of the corresponding commercial enzymes. The multi-enzyme saccharification reaction can be performed simultaneously with the ethanol fermentation process using a thermotolerant yeast Candida tropicalis BCC7755. The combined process produced 14.3 g/l ethanol from 4% (w/v) cassava pulp after 30 h of fermentation. The productivity rate of 0.48 g/l/h is equivalent to 93.7% of the theoretical yield based on total starch and cellulose, or 85.4% based on total fermentable sugars. The non-thermal enzymatic saccharification process described is more energy efficient and yields more fermentable sugar than the conventional enzymatic process. Furthermore, the process is applicable for production of various bio-products of economic importance. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.