The increasing demand and expectations for successful commercialization of second-generation bio-ethanol drives the industry’s continual development to answer this call. Genencor has continued to lead the development of highly effective yet low cost enzyme complexes to provide an economical and environmentally-friendly solution to bio-based fuel production. Accellerase® biomass enzymes allow for hydrolysis of cellulose and hemicellulose to fermentable sugars. In this work, cellulase and hemicellulase activities were characterized to understand the effects of varying process parameters. In particular, both temperature and pH optima of Accellerase® biomass enzyme were determined. The latest results of enzymatic performance and the potential bio-ethanol production from lignocellulosic biomass will be presented under different process configurations.