Effect of pore size in substrate and diffusion of enzyme on hydrolysis of cellulosic materials with cellulases

Abstract

The effect of cellulase size on hydrolysis was studied by comparing the behavior of crosslinked cellulase (CC) with normal cellulase (FC). The average molecular weight of the CC was at least three times the molecular weight of the FC. The amounts of each enzyme were adjusted so that the degree of solubilization after 2 h was the same. The degree of solubilization of Avicel with CC was higher than that with FC in the late stage of reaction. The degree of solubilization of pretreated lignocelluloses was much greater than that of Avicel, but the degree of solubilization with CC was lower than that with FC at all times during the reaction. The degree of solubilization of artificial lignified Avicel was higher with FC than with CC, but the degree of solubilization of de-lignified the artificial lignified Avicel was lower with FC than with CC. The degree of solubilization of amorphous cellulose with FC was the same as that with CC at all times during the reaction. These behaviors are examined by the hypothesis that when small pores dominate, the smaller enzyme components diffuse into the pores and become inactive since synergism with the larger components is no longer possible, whereas, when larger pores dominate, the entire enzyme can diffuse in and therefore the available surface area is increased. This hypothesis is supported by direct measurement of the pore size in two of the substrates and by diffusion inside Avicel of only smaller molecular cellulase component.

1 H NMR investigation of water accessibility in cellulose of pretreated sugarcane bagasse

“…Among the various factors that affect the rate of enzymatic digestibility, the modification of sample porosity was identified as one of the most important, because it directly influences the enzyme access to the substrate [6,26,27]. Due to the intimate contact between the cellulose and the enzymes that is required for the hydrolytic action to take place, the overall surface area, hydrated and accessible to the enzyme action, assumes a fundamental role to the process efficiency.…”

“…These techniques may use probing molecules, such as dextran in solute exclusion methods, or water in differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) relaxometry [27][28][29][30]. They can also be based on the adsorption of a given molecule to lignocellulosic substrates, for instance, nitrogen adsorption to pore surfaces (BET method), proteins and enzymes adsorption, and the adsorption of dyes with a specific affinity to cellulose domains (Simon's staining method) [6,26,31].…”

“…Tanaka et al [26] compared the efficiency of enzymes with different sizes (cross-linked or normal cellulases) to deconstruct microcrystalline and amorphous cellulose. They observed that synergetic effects and hydrolysis yields are favored by the presence of pores on the substrate which are sufficiently large to allow the enzyme to diffuse in.…”

Tools for the Characterization of Biomass at the Nanometer Scale

“…Despite the shortcoming of the solute exclusion technique, it is very useful for under standing the effects of molecular size on accessibility (26,27). In addition, a very useful pore structure model is based on the results of the method (10).…”

Identification and molecular modeling of a family 5 endocellulase from Thermus caldophilus GK24, a cellulolytic strain of Thermus thermophilus

“…It is degraded by enzymes produced by both fungi and bacteria. As cellulose is a very stable polymer, effective hydrolysis of it requires the synergistic action of several enzymes, including endo-β-1,4-glucanases, exo-β-1,4-glucanases (or cellobiohydrolase) and β-glucosidases [9][10][11][12][13][14][15][16][17][18][19][20][21][22]. The mechanism of cellulose hydrolysis by cellulases has been studied extensively [23][24][25][26].…”