Hydrolysis of microcrystalline cellulose by cellobiohydrolase I and endoglucanase II from Trichoderma 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 degrees C) and enzyme loading (0.16 micromol/g Avicel). In isotherm studies the enzyme loading was varied in the range of 0.08-2.56 micromol/g at 4 degrees 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 degrees 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 degrees 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.

A structural study of Hypocrea jecorina Cel5A

“…The deeper cleft contains a hydrophobic patch (F14, V27, Y28, Y40, F34, W292, A294, F297, Y301) surrounded by the b1-a1 loop (residues [15][16][17][18][19][20][21][22], the sidechain of W185, residues 104-107, residues 146-150, and the b6-a6 loop (residues 225-229) [ Fig. 1(C)].…”

“…Previous studies characterize Hj_Cel5A as a promiscuous enzyme that generates a wide range of products including glucose, cellobiose, and cellotriose. 16 The noncatalytic binding groove appears more hydrophilic and shallower than that of Ta_Cel5A. Further testing may reveal whether product inhomogeneity results from scant interaction between Hj_Cel5A and the reducing end of the chain beyond the active site.…”

Substrate Recognition and Hydrolysis by a Family 50 exo-β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans

“…Davies and Henrissat (25) forwarded the observation that enzymes possessing a tunnel-shaped catalytic groove typically act as processive enzymes. Processivity has been described for several types of glycoside hydrolases, including amylases, ␤-glucanases, cellulases, chitinases, carrragenases, and polygalacturonases (57)(58)(59)(60)(61)(62). The particular topology of such a catalytic site has been shown to be a key factor in the efficiency of microcrystalline polysaccharide degradation (63)(64)(65).…”

The Development and Use of an Elisa-Based Method to Follow the Distribution of Cellulase Monocomponents During the Hydrolysis of Pretreated Corn Stover

“…The cellulase monocomponents Cel7A, Cel6A, Cel7B, and Cel5A were purified from Celluclast (Novozyme) using previously described methods [12,[30][31][32]. The Ninhydrin assay [33] was then used to determine the concentrations of these purified enzymes as well as the commercial enzyme mixtures.…”

“…Unfortunately, many of these model substrates are not specific enough to distinguish individual enzymes. Protein chromatography techniques have also been utilized to fractionate the enzyme mixture down to its individual components [11,12]. However, this approach is laborious and, depending on the purification protocols used, the enzyme mixture may not always completely separate into its individual components [13].…”