Kinetics and Modeling of Disproportionation Reactions Catalyzed by Clostridium Acetobutylicum and Ruminococcus Flavefaciens Endoglucanases

Cellotetraose, cellopentaose, and cellohexaose were separately incubated with a Clostridium acetobutylicum glycoside hydrolase family 44 endoglucanase, and the resulting reactant and product concentrations were obtained by capillary electrophoresis chromatography. Cellotetraose was slowly converted to more cellotriose than glucose, with no cellobiose being formed and with most of the cellotetraose remaining at long incubation times. Cellopentaose was hydrolyzed more quickly to cellotetraose, cellobiose, cellotriose, and glucose, in order of near-equilibrium concentrations, with a very small amount of cellopentaose remaining. Cellohexaose was even more quickly converted to cellotetraose, cellobiose, cellopentaose, cellotriose, and glucose, with cellopentaose concentration decreasing after reaching a peak. No cellohexaose remained after a short time. A kinetic model, including both hydrolysis and transglycosylation reactions yielding products larger than the substrates, was formulated based on these results and on previously reported results from a Ruminococcus flavefaciens glycoside hydrolase family 44 endoglucanase, each set showing asymmetric attack on substrates as well as product dispropor­tionation. The model fit data obtained for digestion of the three substrates by C. acetobutylicum endoglucanase and by R. flavefaciens endoglucanase with just a catalytic domain. It fit data obtained for the R. flavefaciens endoglucanase with a catalytic domain/carbohydrate binding module complex somewhat less well. It appears that the absence of observed larger transglycos­ylation products in capillary electrophoresis chromatographs is due to product insolubility or to concentrations near or below the detection limit.