There are several different kinds of cellulases, and they differ mechanisti­cally and structurally. Each cellulolytic microbial group has an enzyme sys­tem unique to it. The enzyme capabilities range from those with which only soluble derivatives of cellulose can be hydrolyzed to those with which a cel­lulose complex can be disrupted. Although it is a usual practice to refer to a mixture of compounds that have the ability to degrade cellulose as cellulase, it is actually composed of a number of distinctive enzymes. Based on the specific type of reaction catalyzed, the cellulases may be characterized into five general groups, namely,

1. Endocellulase cleaves internal bonds to disrupt the crystalline struc­ture of cellulose and expose individual cellulose polysaccharide chains.

2. Exocellulase detaches two or four saccharide units from the ends of the exposed chains produced by endocellulase, resulting in disac­charides or tetrasaccharides, such as cellobiose. Cellobiose is a disac­charide with the formula [HOCH2CHO(CHOH)3]2O. There are two principal types of exocellulases, or cellobiohydrolases (CBH): (a) CBH-I works processively from the reducing end of cellulose and (b) CBH-II works processively from the nonreducing end of cellulose.

In this description, processivity is the ability of an enzyme to con­tinue repetitively its catalytic function without dissociating from its substrate. By an active enzyme being held onto the surface of a solid substrate, the chance for reaction is significantly enhanced.

3. Beta-glucosidase or cellobiase hydrolyzes the exocellulase products, disaccharides and tetrasaccharides, into individual monosaccharides.

4. Oxidative cellulases depolymerize and break down cellulose mole­cules by radical reactions, as in the case with a cellobiose dehydro­genase (acceptor), which is an enzyme that catalyzes the chemical reaction of

cellobiose + acceptor ^ cellobiono-1,5-lactone + reduced acceptor by which cellobiose is dehydrogenated and the acceptor is reduced.

5. Cellulose phosphorylases depolymerize cellulose using phosphates instead of water.

In most cases, the enzyme complex breaks down cellulose to beta-glu­cose. This type of cellulase enzyme is produced mainly by symbiotic bac­teria. Symbiotic bacteria are bacteria living in symbiosis (close and long-term interaction) with another organism or each other. Enzymes that hydro­lyze hemicellulose are usually referred to as hemicellulase and are still com­monly classified under cellulases. Enzymes that break down lignin are not classified as cellulase, strictly speaking. Along with diverse types of enzymes, it must be clearly pointed out that a principal challenge in hydrolytic degradation of biomass into fermentable sugars is how to make these different enzymes work together as a synergistic enzyme system. For example, cellulases and hemicellulases are secreted from a cell as free enzymes or extracellular cellulosomes (complexes of cellulolytic enzymes created by bacteria). The collective activity of these enzymes in a system is likely to be more active than, or at least quite different from, the individual activity of an isolated enzyme.

The enzymes described above can be classified into two types: progressive (also known as processive) and nonprogressive (or, nonprocessive) types. Progressive cellulase will continue to interact with a single polysaccharide strand, whereas nonprogressive cellulase will interact once, disengage, and then engage another polysaccharide strand.

Based on the enzymatic capability, cellulase is characterized into two groups, namely, Q enzyme or factor and CX enzyme or factor [41]. The Q factor is regarded as an "affinity" or prehydrolysis factor that transforms highly ordered (crystalline) cellulose, (i. e., cotton fibers or Avicel) into linear and hydroglucose chains. The Q factor has little effect on soluble deriva­tives. Raw cotton is composed of 91% pure cellulose. As such, it serves as an essential precursor to the action of the CX factor. The CX (hydrolytic) factor breaks down the linear chains into soluble carbohydrates, usually cellobiose (a disaccharide) and glucose (a monosaccharide).

Microbes rich in Q are more useful in the production of glucose from the cellulose. Moreover, because the Q phase proceeds more slowly than the subsequent step, it is the rate controlling step. Among the many microbes, Trichoderma reesei surpasses all others in the possession of Q complex. Trichoderma reesei is an industrially important cellulolytic filamentous fun­gus and is capable of secreting large amounts of cellulases and hemicel — lulases [44]. Recent advances in cellulase enzymology, cellulose hydrolysis (cellulolysis), strain enhancement, molecular cloning, and process design and engineering are bringing T. reesei cellulases closer to being a commer­cially viable option for cellulose hydrolysis [45]. The site of action of cellulo­lytic enzymes is important in the design of hydrolytic systems (CX factor). If the enzyme is within the cell mass, the material to be reacted must diffuse into the cell mass. Therefore, the enzymatic hydrolysis of cellulose usually takes place extracellularly, where the enzyme is diffused from the cell mass into the external medium.

Another important factor in the enzymatic reaction is whether the enzyme is adaptive or constitutive. A constitutive enzyme is present in a cell at all times. Adaptive enzymes are found only in the presence of a given substance, and the synthesis of the enzyme is triggered by an inducing agent. Most of the fungal cellulases are adaptive [15, 41].

Cellobiose is an inducing agent with respect to Trichoderma reesei. In fact, depending on the circumstances, cellobiose can be either an inhibitor or an inducing agent. It is inhibitory when its concentration exceeds 0.5 to 1.0%. Cellobiose is an intermediate product and is generally present in concentra­tions low enough to permit it to serve as a continuous inducer [46].

A milestone achievement (2004) accomplished by the National Renewable Energy Laboratory in collaboration with Genencor International and Novozyme Biotech is of significance in making effective cellulase enzymes at substantially reduced costs, as mentioned in an earlier section.

4.5.2 Enzymatic Processes

All enzymatic processes basically consist of four major steps that may be combined in a variety of ways: pretreatment, enzyme production, hydroly­sis, and fermentation, as represented in Figure 4.7.

4.5.3.1 Pretreatment

It has long been recognized that some form of pretreatment is necessary to achieve reasonable rates and yields in the enzymatic hydrolysis of biomass. Pretreatment has generally been practiced to reduce the crystallinity of cel­lulose, to lessen the average degree of polymerization of the cellulose and the lignin-hemicellulose sheath that surrounds the cellulose, and to allevi­ate the lack of available surface area for the enzymes to attack. A typical pretreatment system consists of size reduction, pressure sealing, heating, reaction, pressure release, surface area increase, and hydrolyzate/solids sep­aration [47].

Mechanical pretreatments such as intensive ball milling and roll milling have been investigated as a means of increasing the surface area, but they require exorbitant amounts of energy. The efficiency of the chemical process can be understood by considering the interaction between the enzymes and the substrate. The hydrolysis of cellulose into sugars and other oligomers is a solid phase reaction in which the enzymes must bind to the surface to catalyze the reaction. Cellulase enzymes are large proteins, with molecular weights ranging from 30,000 to 60,000 and are thought to be ellipsoidal with major and minor dimensions of 30 to 200 A°. The internal surface area of wood is very large, however, only about 20% of the pore volume is accessible to cellulase-sized molecules. By breaking down the tight hemicellulose-lig — nin matrix, hemicellulose or lignin can be separated and the accessible vol­ume can be greatly increased. This removal of material greatly enhances the enzymatic digestibility.

The hemicellulose-lignin sheath can be disrupted by either acidic or basic catalysts. Basic catalysts simultaneously remove both lignin and hemicel- lulose, but suffer large consumption of the base through neutralization by ash and acid groups in the hemicellulose. In recent years, attention has been focused on the acidic catalysts. They can be mineral acids or organic acids generated in situ by autohydrolysis of hemicellulose.

Various types of pretreatments are used for biomass conversion. The pre­treatments that have been studied in recent years are steam explosion auto­hydrolysis, wet oxidation, organosolv, and rapid steam hydrolysis (RASH). The major objective of most pretreatments is to increase the susceptibility of cellulose and lignocellulose material to acid and enzymatic hydrolysis. Enzymatic hydrolysis is a very sensitive indicator of lignin depolymeriza­tion and cellulose accessibility. Cellulase enzyme systems react very slowly with untreated material; however, if the lignin barrier around the plant cell is partially disrupted, then the rates of enzymatic hydrolysis are increased dramatically.

Most pretreatment approaches are not intended to actually hydrolyze cel­lulose to soluble sugars, but rather to generate a pretreated cellulosic residue that is more readily hydrolyzable by cellulase enzymes than native biomass. Dilute acid hydrolysis processes are currently being proposed for several
near-term commercialization efforts until lower-cost commercial cellulase preparations become available. Such dilute acid hydrolysis processes typi­cally result in no more than 60% yields of glucose from cellulose.