Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.
A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative 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 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-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 1.0 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 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides 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.
Huang
L
L
Department of Microbiology, University of Guelph, Ontario, Canada.
Forsberg
C W
CW
Thomas
D Y
DY
D001439:Bacteroides / Q000201:enzymology*; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D002712:Chlorides; D009844:Oligosaccharides; D002475:Cellobiose; D002482:Cellulose; D006026:Glycoside Hydrolases; C034169:cellobiosidase