Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion. Results: GHs and accessory proteins were produced by representative brown-and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and beta-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-beta-glucosidases, and GH10-xylanases. Conclusions: The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5-and GH45-endoglucanases, GH3-beta-glucosidases, and GH10-xylanases.9Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [08/56256-5, 2014/06923-6]Conselho Nacional de Pesquisa (CNPq) [442333/2014-5, 310186/2014-5, 140796/2013-4]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)