Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S85.
Two endoglucanases designated EG1 and EG2 were purified by column chromatography from the nonsedimentable extracellular culture fluid of Bacteroides succinogenes S85. They accounted for approximately 32 and 11%, respectively, of the total endoglucanase present in the nonsedimentable fraction. The most active enzyme (EG1) had a molecular weight of 65,000, pI of 4.8, and temperature and pH optima of 39 degrees C and 6.4, respectively. The Km for carboxymethyl cellulose was 3.6 mg/ml, and the Vmax was 84 U/mg. The major products of cellulose hydrolysis catalyzed by EG1 were cellotriose and cellobiose. EG2 was present as two components with molecular weights of 118,000 and 94,000. The two components had nearly identical cyanogen bromide peptide maps, thereby indicating that the 94,000-dalton component was a proteolytic degradation product of the 118,000-dalton enzyme. The larger component, which was more abundant in the culture fluid than the smaller form was, had a Km of 12.2 mg/ml and a Vmax of 10.4 U/mg. It was a basic protein with a pI of 9.4, a temperature optimum of 39 degrees C, and a pH optimum of 5.8. The major product of cellulose hydrolysis was cellotetraose. EG2 exhibited specific binding to acid-swollen cellulose, whereas EG1 did not, and neither of them had affinity for crystalline cellulose. Based on the substrate specificities and the affinities of the two enzymes for cellulose, we postulated that EG2 is involved in the early stages of cellulose hydrolysis and that EG1 is active primarily on the products arising from EG2.
McGavin
M
M
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada.
Forsberg
C W
CW
D001439:Bacteroides / Q000201:enzymology*; D002480:Cellulase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D010449:Peptide Mapping; D013379:Substrate Specificity; D013696:Temperature
D002482:Cellulose; C056374:endoglucanase 1; C056373:endoglucanase 2; D002480:Cellulase