A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gramnegative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39°C and pH 6.5, the activity of the enzyme with p-nitrophenyli--D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.o mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45°C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl -cellulose.Of the cellulolytic rumen bacteria, Bacteroides succinogenes has been described as a predominant cellulose degrader (8, 24). Its cellulase system has been the subject of extensive research in the past few years (16, 19-21, 23, 32, 37, 40). Both endoglucanase and cellobiase activities have been demonstrated in this bacterium for some time (19)(20)(21)37). Recently, two endoglucanases were purified from the extracellular-culture fluid of B. succinogenes (32). These two enzymes hydrolyzed cellooligosaccharides and amorphous cellulose but produced different end products. Examination of the exo-type cellulase activities in B. succinogenes has also been carried out. In a previous study, we identified an exo-type cellodextrinase in the periplasm of B. succinogenes cells grown on cellulose (23). This enzyme was capable of the successive removal of terminal cellobiose units from water-soluble cellodextrins, but not from longer cellulose chains, and therefore appeared to be different from the well-characterized fungal exoglucanases. We therefore decided to undertake further studies on exo-type cellulase activity.Here we report on the purification and characterization of a unique cellobiosidase from B. succinogenes, the activity of which was significantly enhanced by chloride and several other anions. This cellobiosidase may be involved in the initial steps of cellulose hydrolysis.
MATERIALS AND METHODSOrganism and growth conditions. B. succinogenes S85 (previously obtained from M. P. Bryant, University of Illinois, Urbana) was grown continuously in a Multigen bench top fermentor (Multigen; New Brunswick Scientific Co., Inc., Edison, N.J.) equipped with a 2-liter chemstat vessel (C-30) and an automatic pH control, as described previously (23). The chemostat medium use...