Characterization of a b-Glucosidase Produced by a High-Specific Growth-Rate Mutant of Cellulomonas flavigena Gaspar A. Barrera-Islas, 1 Ana C. Ramos-Valdivia, 1 Luis M. Salgado, 2 Teresa Ponce-Noyola 1 1 Departamento de Biotecnología y Bioingeniería; 2 Departamento de Bioquímica, CINVESTAV-IPN, Avenida IPN 2508, Zacatenco, MØxico, 07000 DF, MØxico Received: 6 March 2006 / Accepted: 18 May 2006 Abstract. The mutant strain PN-120 of Cellulomonas flavigena produces a ß-glucosidase that is 10-fold more active than the corresponding enzyme isolated from the parental strain. These enzymes were partially purified through Q Sepharose and Bio-Gel filtration. A single protein band was detected on polyacrylamide–gel electrophoresis/zymogram using 4-methylumbelliferyl-b-D-glucoside. On sodium dodecyl sulfate–PAGE, the enzyme displayed three protein bands, suggesting that in C. flavigena the enzyme is oligomeric with a molecular mass of 210 kDa. On purification, the specific activity of ß-glucosidase isolated from PN-120 was increased 16-fold and showed three times more affinity for cellobiose than the enzyme of the parental strain; nevertheless, the optimum pH and temperature were similar for both enzymes. The kinetic parameters suggested that the increase in the activity of the enzyme, from the mutant strain, was caused by a mutation that affects the catalytic site of the enzyme. The partial amino-acid sequence of the isolated enzyme confirmed that it is a b-glucosidase because of its homology with other b-glucosidases produced by cellulolytic bacteria and fungi. The use of cellulosic residues available every year as a source of glucose to produce ethanol and other fuel products is creating major interest because they represent an alternative source of energy [1, 2]. The degradation of the cellulose involves the action of an enzymatic complex consisting of at least three types of enzymes where the b-glucosidase (EC3.2.1.21) hydrolyzes the last step, i.e., turning cellobiose into glucose [3]. Most of the b-gluco- sidases are inhibited by glucose accumulation during the reaction, thus limiting the cellulose degradation rate [4]. Two alternatives to solve this problem can be used: (1) the addition of an external b-glucosidase to commercial cellulase preparations [5, 6] or (2) finding a suitable b-glucosidase produced by micro-organisms through strain selection with improved catalytic properties. C. flavigena is an excellent source of cellulose degrading enzymes because it is able to grow using dif- ferent types of agriculture wastes as carbon source [7]. A mutant strain (PN-120) was isolated that had 10-fold more b-glucosidase activity than the wild-type strain when both strains were grown on sugar cane bagasse [8]. To characterize the enzyme and to gain insight into the reason for the high activity in the mutant strain, we performed partial purification and characterization of the b-glucosidase produced from the wild-type strain and its mutant, PN-120, when both strains were grown on sugar cane bagasse as carbon source. Materials and Methods Micro-organisms and culture conditions. C. flavigena wild-type (CDBB531) strain and its mutant PN-120 stain [8] were used in all experiments as source of b-glucosidase. Cells were grown in Erlenmeyer flasks containing saline medium supplemented with biotin (10 lgÆl )1 ), thiamine (1 mgÆl )1 ), and 1% w/v alkali-pretreated sugar cane bagasse in an orbital shaker at 100 rpm (New Brunswick Science, Edison, NJ) at 37°C for 48 hours. Production and purification of b-glucosidase. The strains of C. flavigena, wild-type strain, and mutant PN-120 strain were grown in 2-l Erlenmeyer flasks, and the procedures were carried out in parallel as described later. The residual substrate was removed by Correspondence to: T. Ponce-Noyola; email: tponce@cinvestav.mx CURRENT MICROBIOLOGY Vol. 54 (2007), pp. 266–270 DOI: 10.1007/s00284-006-0105-7 Current Microbiology An International Journal ª Springer Science+Business Media, LLC 2007