Hemicellulose Hydrolysis Mechanisms
Due to the heterogeneous nature of hemicellulose a series of enzymes are involved in the saccharification and complete hydrolysis and requires the action of a whole enzyme system. This system is usually composed of P-xylanase, в-xylosidase, and enzymes such as a-L-arabinofuranosidase, a-glucuronidase, acetyl xylan esterase, and hydroxycinnamic acid esterases that cleave side chain residues from the backbone. All these enzymes act cooperatively to convert hemicellulose to its constituents. The two major glycosyl hydrolases depolymerizing the backbone of hemicellulose are endo-1,4-^-D-xylanase and endo-1,4-^-D-mannanase. The enzymes endo-1,4-^-xylanases cleave the glycosidic bonds in the linear segments of the chain with ^-1,4-xylan links, bringing about a reduction in the degree of polymerization of the substrate. In this degradation, xylan backbone is not attacked randomly, but the bonds selected for hydrolysis depend on the nature of the substrate molecule, i. e., on the chain length, the degree of branching, and the nature of the substituents [65]. In the initial stage of this main hydrolysis, products are в-D-xylopyranosyl oligomers, but at a later stage, small molecules such as mono-, di-, and trisaccharides of в-D-xylopyranosyl may be produced [66]. Cellulase enzymes act on the common hemicellulose components xylan, galacto — glucomannan, and a-1,5-L-arabinan, and their action points are
shown in Figure 6.7. In addition to this, cellulase enzyme mixture contains disaccharide hydrolyzing enzymes P-xylosidase and P-mannosidase for the hydrolysis of xylobiose and mannobiose as shown in Figure 6.7.
Xylosidases are essential for the complete breakdown of xylan as they hydrolyze xylooligosaccharides to xylose [67]. The enzymes arabinosidase, a-glucuronidase and acetyl xylan esterase act in synergy with the xylanases and xylosidases by releasing the substituents on the xylan backbone to achieve a total hydrolysis of xylan to monosaccharides [68]. For example, the main sugar moiety of galac- toglucomannans is D-mannose, but for its complete breakdown into simple sugars, the synergistic action of endo-1,4-P-mannanases (EC 3.2.1.78) and exo acting P-mannosidases (EC 3.2.1.25) is required to
cleave the polymer backbone. In addition, supplementary enzymes such as в-glucosidases (EC 3.2.1.21), a-galactosidases (EC 3.2.1.22) and acetyl mannan esterases are required to remove side chain sugars that are attached at various points on mannans [69]. A number of endoglucanases have been reported to hydrolyze xyloglucan as a substrate analog, however, few endo-^-1,4-glucanases have high activity toward xyloglucan, with little or no activity towards cellulose or cellulose derivatives [70, 71]. Among these enzyme families, xyloglucanases are known to have high specific activity towards xyloglucan, with inversion of the anomeric configuration, and both endo — and exo-type hydrolases have been found in several microorganisms [71]. The exo-type enzymes recognize the reducing end of xyloglucan oligosaccharide (oligoxyloglucan reducing-end-specific cellobiohydrolase, EC 3.2.1.150, from Geotrichum sp. M128 [71] and oligoxyloglucan reducing — end-specific xyloglucanobiohydrolase from Aspergillus nidulans [72]), whereas the endo-type enzymes hydrolyze xyloglucan polymer randomly.
Most of the commercial cellulase preparations contain hemi — cellulases as well; however there are many reports on isolation of cellulase-free pure hemicellulose degrading enzymes and specific activity measurements and characterizations. Using the correct proportion of hemicellulases in the saccharification enzyme mixture is important, since hemicellulases facilitate cellulose hydrolysis by exposing the cellulose fibers, thus making them more accessible. Biochemistry and industrial applications of hemicellulases are discussed in a number of good review articles [73-75,66].