AbstractThe ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and β‐glucosidase activity. Although the well‐documented relief of product inhibition by β‐glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non‐cellulosic polysaccharides. It is suggested that so‐called “accessory” enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non‐cellulosic polysaccharides that coat cellulose fibers. A high‐throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a ∼twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis. Biotechnol. Bioeng. 2007;97: 287–296. © 2006 Wiley Periodicals, Inc.