The overall hydrolysis is based on the synergistic action of three distinct cel — lulase enzymes and is dependent on the concentration ratio and the adsorp­tion ratio of the component enzymes: endo-p-glucanases, exo-p-glucanases, and p-glucosidases. Endo-p-glucanases attack the interior of the cellulose polymer in a random fashion [43], exposing new chain ends. Because this enzyme catalyzes a solid phase reaction, it adsorbs strongly but reversibly to the microcrystalline cellulose (also known as Avicel). The strength of the adsorption is greater at lower temperatures. This enzyme is necessary for the hydrolysis of crystalline substrates. The hydrolysis of cellulose results in a considerable accumulation of reducing sugars, mainly cellobiose, because the extracellular cellulase complex does not possess cellobiose activity. Sugars that contain aldehyde groups that are oxidized to carboxylic acids are classified as reducing sugars.

Exo-p-glucanases remove cellobiose units (which are disaccharides with the formula ([HOCH2CHO(CHOH)3]2O) from the nonreducing ends of cel­lulose chains. This is also a solid-phase reaction, and the exo-p-glucanases adsorb strongly on both crystalline and amorphous substrates. The mecha­nism of the reaction is complicated because there are two distinct forms of both endo — and exo-enzymes, each with a different type of synergism with the other members of the complex. As these enzymes continue to split off cellobiose units, the concentration of cellobiose in solution may increase. The action of exo-p-glucanases may be severely inhibited or even stopped by the accumulation of cellobiose in the solution.

The cellobiose is hydrolyzed to glucose by the action of p-glucosidase. Glucosidase is any enzyme that catalyzes hydrolysis of glucoside. P-Glucosidase catalyzes the hydrolysis of terminal, nonreducing beta-D — glucose residues with release of beta-D-glucose. The effect of p-glucosidase on the ability of the cellulase complex to degrade Avicel has been investi­gated by Kadam and Demain [73]. They determined the substrate specific­ity of the p-glucosidase and demonstrated that its addition to the cellulase complex enhances the hydrolysis of Avicel, specifically by removing the accumulated cellobiose. A thermostable p-glucosidase form, clostridium thermocellum, which is expressed in Escherichia coli, was used to deter­mine the substrate specificity of the enzyme. The hydrolysis of cellobi — ose to glucose is a liquid-phase reaction and p-glucosidase adsorbs either quickly or not at all on cellulosic substrates. p-Glucosidase’s action can be slowed or halted by the inhibitive action of glucose accumulated in the solution. The accumulation may also induce the entire hydrolysis to a halt as inhibition of the p-glucosidase results in a buildup of cellobiose, which in turn inhibits the action of exo-glucanases. The hydrolysis of the cel — lulosic materials depends on the presence of all three enzymes in proper amounts. If any one of these enzymes is present in less than the required amount, the other enzymes will be inhibited or lack the necessary sub­strates upon which to act.

The hydrolysis rate generally increases with increasing temperature. However, because the catalytic activity of an enzyme is also related to its shape, the deformation of the enzyme at high temperature can inactivate or destroy the enzyme. To strike a balance between increased activity and increased deactivation, it is preferable to run fungal enzymatic hydrolysis at approximately 40-50°C.

Although enzymatic hydrolysis is preferably carried out at a low tempera­ture of 40-50°C, dilute acid hydrolysis is carried out at a substantially higher temperature. Researchers at the National Renewable Energy Laboratory (NREL) reported results for a dilute acid hydrolysis of softwoods in which the conditions of the reactors were as follows [74].

1. Stage 1: 0.7% sulfuric acid, 190°C, and a 3-min residence time

2. Stage 2: 0.4% sulfuric acid, 215°C, and a 3-min residence time

Their bench-scale tests also confirmed the potential to achieve yields of 89% for mannose, 82% for galactose, and 50% for glucose, respectively. Fermentation with Saccharomyces cerevisiae achieved ethanol conversion of 90% of the theoretical yield [75].