1.4.1 Cellulose and cellulose degradation enzymes
Life on Earth depends on photosynthesis, which results in production of plant biomass.
Cellulose, the most abundant component of plant biomass, is found in nature almost exclusively in plant cell walls, although it can be produced by some animals (e.g., tunicates) and a few bacteria. In
a few cases (notably cotton bolls), cellulose is present in a nearly pure state. In most cases, the cellulose fibers are embedded in a matrix of other structural biopolymers, primarily hemicelluloses and lignin, which comprise 20 to 35 and 5 to 30% of the plant dry weight (Lynd et al., 1999;
Sjostrom, 1993). Chemically, cellulose is a linear condensation polymer consisting of D-anhydro-glucopyranose joined together by β-1,4-glycosidic bonds with a degree of polymerization (DP) from 100 to 20,000 (Krassig, 1993; Tomme et al., 1995; Zhang and Lynd, 2004a). Anhydrocellobiose is the repeating unit of cellulose. Coupling of adjacent cellulose chains and sheets of cellulose by hydrogen bonds and van der Waal's forces results in a parallel alignment and a crystalline structure with straight, stable supra-molecular fibers of great tensile strength and low accessibility (Demain et al., 2005; Krassig, 1993; Nishiyama et al., 2003; Notley et al., 2004; Zhang and Lynd, 2004b;
Zhbankov, 1992). Therefore, although abundant in nature, cellulose is a particularly difficult polymer to degrade (Mansfield et al., 1999). Degradation of hemicellulose, pectin and lignin is generally easier.
Cellulose molecules can be hydrolysed by a class of enzymes called cellulases, which are produced primarily by fungi, bacteria, protozoa, and some plants and animals (Norkrans, 1963). In micro-organisms, there are two types of enzyme systems for cellulose degradation which have been observed. In the case of aerobic fungi and bacteria, several individual endoglucanases, exoglucanases and ancillary enzymes, are secreted which, together, can work synergistically to hydrolyze cellulose, therefore called non-complexed systems. In anaerobic microorganisms, a different type of system has evolved that involves the formation of a large, extracellular enzyme complex called the cellulosome (or complexed system). The cellulosome consists of scaffolding proteins and many bound cellulosomal enzymes (Bayer et al., 1985). The scaffolding proteins are large nonenzymatic proteins that usually contain a number of cohesin domains (Coh) and cellulose binding domains. However, hydrophilic domains, dockerin II domains, the enzyme coding domain, and a number of unidentified domains whose functions remain unknown have also been observed in some of the scaffolding proteins (Doi et al., 2003). The structure and function of cellulosomes have been reviewed (Doi et al., 2003; Doi and Kosugi, 2004; Bayer et al., 1985; Bayer et al., 2004).
Fig 1.13 The hydrolysis of amorphous and microcrystalline cellulose by noncomplexed (A) and complexed (B) cellulase systems. The solid squares represent reducing ends, and the open squares represent nonreducing ends. Amorphous and crystalline regions are indicated.
Cellulose, enzymes, and hydrolytic products are not shown to scale (Lynd et al., 2002).
Components of cellulase systems were first classified based on their mode of catalytic action and have been more recently classified based on structural properties (Henrissat et al., 1998). Three major types of enzymatic activities are found: (i) endoglucanases or 1,4-ß-D-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii) exoglucanases, including 1,4-ß-D-glucan 1,4-ß-D-glucan-4-glucanohydrolases (also known as cellodextrinases) (EC 3.2.1.74) and 1,4-ß-D-glucan cellobiohydrolases (cellobiohydrolases) (EC 3.2.1.91), and (iii) ß-glucosidases or ß-glucoside glucohydrolases (EC 3.2.1.21). Endoglucanases catalyze randomly at internal amorphous sites in the cellulose polysaccharide chain, generating oligosaccharides of various lengths and consequently new chain ends. Exoglucanases act in a processive manner on the reducing or nonreducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose,
presumably peeling cellulose chains from the microcrystalline structure (Teeri, 1997). ß-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose (see Fig 1.13).
1.4.2 Applications of cellulases
Cellulosic materials are particularly attractive because of their relatively low cost and plentiful supply. Since the early 1980s, biotechnology of cellulases has developed, first in animal feed, later followed by food applications (Voragen, 1992). Later on, cellulases have proven their utilities in the textile industry for cotton softening and denim finishing (Godfrey, 1996), in the detergent market for color care, cleaning, and anti-deposition, in the food industry for mashing, and in the pulp and paper industries for de-inking, improvement, and fiber modification (Godfrey and West, 1996; Bhat, 2000). The cellulase market is expected to expand dramatically when cellulases are used to hydrolyze pretreated cellulosic materials into sugars, which can be fermented to produce commodities such as ethanol and other bio-products on a large scale (Hahn-Hagerdal et al., 2006).
Some of the most promising applications of cellulases are listed in this part.
In food industry, cellulases are used in extraction and clarification of fruit and vegetable juices, production of fruit nectars and purées, and in the extraction of olive oil (Bhat, 2000). They are also used in carotenoid extraction for producing food coloring agents (Cinar, 2005).
The ß-glucanases, especially from Trichoderma, appear to be suitable for the production of high quality beer from poor quality barley, thus show a great benefit in the brewing industry (Galante et al., 1998b). In the early 1980s, it was suggested that Trichoderma ß-glucanase could be successfully used for wine making from grapes infected with Botrytis cinerea (Dubordieu et al., 1981; Villetaz et al., 1984). This fungus generally attacks nearly ripe grapes under conditions of certain temperatures and humidity, and produces a high molecular mass soluble ß-(1,3) glucan with short side chains linked through ß-(1,6) glycosidic bonds, which alleviates several problems during wine filtration. A ß-glucanase from Trichoderma harzianum was also found to be useful for hydrolysis of glucans from yeast, which have caused adverse effects during filtration and clarification of wine (Galante et al., 1998b).
Cellulases have achieved their worldwide success in textile and laundry because of their ability to modify cellulosic fibres in a controlled and desired manner, so as to improve the quality of fabrics. Cellulases have now become the third largest group of enzymes used in textile and laundry industry (Galante et al., 1998a; Galante et al., 1998a). Bio-stoning and bio-polishing are the
best-known current textile applications of cellulases (Belghith et al., 2001). Cellulases are also increasingly used in household washing powders, since they enhance the detergent performance and allow the removal of small, fuzzy fibrils from fabric surfaces and improve the appearance and colour brightness (Uhlig, 1998).
Cellulases have been used for different purposes in the pulp and paper industry. Cellulase and hemicellulase mixtures have been used for the modification of fibre properties for improving drainage, beatability and runnability of the paper mills (Noe et al., 1986; Pommier et al., 1990). The addition of cellulase and hemicellulase after beating is to improve the drainage properties of pulps, which determine the speed of paper mills. A commercial cellulase/hemicellulase preparation, named Pergalase-A40, from Trichoderma has been used by many paper mills around the world for the production of release papers and wood-containing printing papers (Pommier et al., 1990). For de-inking purpose, most of the published literatures are dealt with cellulases and hemicellulases (Bajpai, 1999).
The most important application currently being investigated is the utilization of lignocellulosic wastes for the production of biofuel. Currently, the US and Brazil are leaders in the production of starch/sugar-based fuels from corn and sugarcane crops, respectively. However, starch raw materials will not be sufficient enough to meet increasing demand and are a controversial resource for bioconversion (Greene et al., 2004; Maki et al., 2009). With the increasing demands for energy and the shrinking energy resources, the utilization of plant biomass for the production of biofuel offers a renewable alternative. A potential application of cellulase is the convertion of cellulosic materials to glucose and other fermentable sugars, which in turn can be used as microbiol substrates for the production of single cell proteins or a variety of fermentation products like ethanol. Theoretically, this is all quite possible; however, technologically, it is not an easy task because of various technological gaps. Cellulosic bioconversion is a multi-step process requiring a multi-enzyme complex for efficient bioconversion into fermentable sugars. However, there is no known organism capable of producing all the necessary enzymes in sufficient quantities. In addition, there is a lack of biocatalysts that can work efficiently and inexpensively at high temperatures and/or low pH conditions used in the bioconversion of lignocellulosic material to bioethanol. Moreover, there is a great need for cost-effective fermentation of derived sugars from cellulose and also from hemicellulose (Wyman et al., 2005). Therefore, there is a lot of work ahead for researchers to efficiency use promising resources – plant biomass.