Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II fromTrichoderma reesei: Adsorption, sugar production pattern, and synergism of the enzymes

Abstract

Microcrystalline cellulose (10 g/L Avicel) was hydrolysed by two major cellulases, cellobiohydrolase I (CBH I) and endoglucanase II (EG II), of Trichoderma reesei. Two types of experiments were performed, and in both cases the enzymes were added alone and together, in equimolar mixtures. In time course studies the reaction time was varied between 3 min and 48 h at constant temperature (40°C) and enzyme loading (0.16 μmol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08–2.56 μmol/g at 4°C and 90 min. Adsorption of the enzymes and production of soluble sugars were followed by FPLC and HPLC, respectively. Adsorption started quickly (50% of maximum achieved after 3 min) but was not completed before 60–90 min. For CBH I a linear relationship was observed between the production of soluble sugars and adsorption, showing that the average activity of the bound CBH I molecules does not change with increasing saturation. For EG II the corresponding curve levelled off which is explained by initial hydrolysis of loose ends on Avicel. The enzymes competed for binding sites, binding of EG II was considerably affected by CBH I, especially at high concentration. CBH I produced more soluble sugars than EG II, except at conversions below 1%. At 40°C when the enzymes were added together they produced 27–45% more soluble sugars than the sum of what they produced alone, i.e. synergistic action was observed (the final conversion after 48 h of hydrolysis was 3, 6, and 13% for EG II, CBH I, and their mixture, respectively). At 4°C, on the other hand, when the conversion was below 2.5%, almost no synergism could be observed. Molar proportions of the produced sugars were rather stable for CBH I (11–15%, 82–89%, and <6% for glucose, cellobiose, and cellotriose, respectively), while it varied considerably with both time and enzyme concentration for EG II. The observed stable but high glucose to cellobiose ratio for CBH I indicates that the processivity for this enzyme is not perfect. EG II produced significant amounts of glucose, cellobiose, and cellotriose, which are not the expected products of a typical endoglucanase activity on a solid substrate. We explain this by hypothesizing that EG II may show processivity due to its extended substrate binding site and the presence of its cellulose binding domain. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:621–634, 1998.

A Multistage Process to Enhance Cellobiose Production from Cellulosic Materials

“…According to Andrić et al [28], glucose directly inhibits β-glucosidase, which results in accumulation of cellobiose. The latter is a strong inhibitor of cellobiohydrolases [15]. Cellobiose inhibition study was reported on the commercial enzyme preparation Celluclast® 1.5 L acting on para-nitrophenyl β-D-cellobioside as substrate [29].…”

“…According to Tomme et al [26] and Suurnäkki et al [27], the extracellular enzyme system produced by T. reesei possesses the ability to adsorb onto cellulose through a cellulosebinding domain, except for endoglucanase III and β-glucosidases. Cellulase adsorption is a necessary step prior to hydrolysis [15].…”

“…Efficient cellulosic bioconversion is a complex process and requires the synergistic action of three kinds of enzymes: endoglucanases, exoglucanases, and β-glucosidases [15][16][17]. Endoglucanases act randomly at internal amorphous sites in the cellulose polysaccharide chain, producing oligosaccharides of various lengths and consequently new sites for the exoglucanases.…”

Study on the Decreased Sugar Yield in Enzymatic Hydrolysis of Cellulosic Substrate at High Solid Loading

“…Such data suggest that the binding capacity of cellulase to cellulose may be directly related to glucose release, and this is consistent with the fact that cellulosic substrate is insoluble and cellulase needs to adhere to cellulose to function. These results agree well with some previous observations, showing that binding of cellulase is related to the hydrolysis of cellulose [12,20].…”

Kinetic Study on the Pretreatment and Enzymatic Saccharification of Rice Hull for the Production of Fermentable Sugars

“…A higher yield of reducing sugar was also observed if increasing the enzyme concentration. However, no significant increase was achieved when cellulase concentration was higher than 1.2 g/L, probably due to the saturation of substrate surface [26]. The maximum hydrolysis rate for each enzyme concentration was found at the beginning of the reaction, followed with a gradual decrease as the saccharification was carried on.…”