January 2015  Vol. 25  No. 1 J. Microbiol. Biotechnol. (2015), 25(1), 57–65 http://dx.doi.org/10.4014/jmb.1401.01045 Research Article jmb Review Characterization of β-Glucosidase Produced by the White Rot Fungus Flammulina velutipes Julieta Mallerman * , Leandro Papinutti, and Laura Levin Laboratorio de Micología Experimental, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, PROPLAME-PRHIDEB CONICET, Buenos Aires, Argentina Introduction β-Glucosidases (β-D-glucoside glucohydrolase, E.C. 3.2.1.21), under physiological conditions, catalyze the hydrolysis of β-1,4-glycosidic bonds from the nonreducing termini presented in alkyl- and aryl-β-D-glycosides, as well as different oligosaccharides (containing 2-6 monosaccharides). These enzymes are widespread in nature, occurring in all domains of living organisms, Archaea, Eubacteria, and Eukaryotes, in which they play varied functions [39]. They represent an important group of enzymes because of their potential uses in various biotechnological processes, including biomass degradation [44], the production of fuel ethanol from cellulosic agricultural residues [22], release of aromatic compounds in the flavor industry [13], and synthesis of useful β-glucosides [24]. Cellulose is the most abundant polymer on Earth, and its complete degradation is accomplished by a cellulolytic complex, which involves the synergistic action of three enzymes: 1,4-β-D-endoglucanase (E.C. 3.2.1.4), 1,4-β-D- cellobiohydrolase (CBH, E.C. 3.2.1.91), and β-glucosidase. It is generally accepted that the endoglucanases and the CBHs act cooperatively and synergistically in depolymerizing cellulose to cellobiose and oligosaccharides, which are then converted by β-glucosidase to glucose [3]. The deficiency in β-glucosidase activity causes the accumulation of the disaccharide cellobiose, leading to the repression of enzyme biosynthesis and end-product inhibition of the upstream enzymes, which result in a limited hydrolysis yield [44]. Therefore, commercially available cellulolytic preparations are often supplemented with β-glucosidase to boost the overall activity, such as that prepared from Trichoderma reesei cellulases [8, 41]. β-Glucosidase is the rate-limiting factor in the conversion of cellulose to glucose for the subsequent production of fuel ethanol. Product inhibition and thermal inactivation of β-glucosidase constitute two major barriers to the development of enzymatic hydrolysis of cellulose as a commercial process. There is an Received: January 24, 2014 Revised: July 4, 2014 Accepted: September 4, 2014 First published online September 4, 2014 *Corresponding author Phone: +54-11-4576-3300; Fax: +54-11-4576-3384; E-mail: julietamllr@gmail.com pISSN 1017-7825, eISSN 1738-8872 Copyright © 2015 by The Korean Society for Microbiology and Biotechnology β-Glucosidase production by the white rot fungus Flammulina velutipes CFK 3111 was evaluated using different carbon and nitrogen sources under submerged fermentation. Maximal extracellular enzyme production was 1.6 U/ml, corresponding to a culture grown in sucrose 40 g/l and asparagine 10 g/l. High production yield was also obtained with glucose 10 g/l and asparagine 4 g/l medium (0.5 U/ml). Parameters affecting the enzyme activity were studied using p-nitrophenyl-β-D-glucopyranoside as the substrate. Optimal activity was found at 50°C and pHs 5.0 to 6.0. Under these conditions, β-glucosidase retained 25% of its initial activity after 12 h of incubation and exhibited a half-life of 5 h. The addition of MgCl 2 , urea, and ethanol enhanced the β-glucosidase activity up to 47%, whereas FeCl 2 , CuSO 4 , Cd(NO 3 ) 2 , and cetyltrimethylammonium bromide inflicted a strong inhibitory effect. Glucose and cellobiose also showed an inhibitory effect on the β-glucosidase activity in a concentration-dependent manner. The enzyme had an estimated molecular mass of 75 kDa. To the best of our knowledge, F. velutipes CFK 3111 β-glucosidase production is amongst the highest reported to date, in a basidiomycetous fungus. Keywords: β-Glucosidase, Flammulina velutipes, submerged fermentation