de Lourdes teixeira de Moraes polizeli 1,2 ✉ β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. they catalyze the ratelimiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for secondgeneration ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 β-glucosidases. Under physiological conditions the β-glucosidases (Bgls-EC 3.2.1.21) catalyze the hydrolysis of β-1,4-glycosidic bonds at the non-reducing termini in alkyl-and aryl-β-D-glycosides, as well as in oligosaccharides containing 2-6 monosaccharides 1-4. The Bgls are ubiquitous in nature and due to their wide range of substrate specificities, the Bgls play various biological roles 5 , and this diversity leads them to be considered as industrially important enzymes 6. In cellulolytic microorganisms, the Bgls may act as cellulolytic enzymes that synergistically function by converting cellulose to glucose. Together with endoglucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), β-glucosidases are involved in the degradation of cellulosic biomass 6-9. Although the activities of cellobiohydrolases and endoglucanases are inhibited by the reaction product (cellobiose), Bgls can overcome this inhibition by the hydrolysis of cellobiose 10-12. A deficiency in Bgls activity can result in the accumulation of cellobiose, leading not only to enzyme inhibition of the upstream enzymes, but also to the repression of enzyme biosynthesis, which results in limitations on hydrolysis yield 12-14. Thus, Bgls can be considered to be the rate-limiting factor in the conversion of cellulose to glucose in biomass saccharification. Due to this important role, there is an increasing demand for the identification, production and characterization of new Bgls that retain their catalytic activity in the presence of glucose 15. Malbranchea pulchella is a thermophilic fungus found in fragments of decomposing plants or cellulose-containing material and is a good producer of trehalases 16 , xylanases 17,18 and β-glucosidases 19 , and it may be considered promising for the production of enzymes of biotechnological interest. The objective of this study was the isolation of a glucose-tolerant GH3 β-glucosidase produced by M. pulchella together with the biochemical char...