Enzymes and Low Molecular Agents
In view of the historical development of the research on wood degration by fungi, this chapter starts with the enzymes invoiveti in the de woody cell Null, although it is now commonly accepted that non-enzymatic low molecular weight metabolites are involved as precursors and / or co-agentswith enzymatic cell wall degradation.
Under the conditions within microbial cells, namely an aqueous environment with pH values around 6 and temperatures of I -50"C, most reactions would run off only very slowly. Enzymes reduce the amount of the necessary activation energy as biocatalysts and control the reaction by substrate and effect specificity.
More than 3,000 enzymes are described. Comparable with the lock/key principle, enzymes possess an active center, into which the substrate must fit, and which thus controls the conversion of the correct substrate (substrate specificity). The protein portion of the enzyme decides on the way of the reaction (effect specificity). Enzymes may consist only of protein or contain additional cofactors (e.g., Mg2+, Mn2+ ) or coenzymes (e.g., vitamin B1). Before the conversion of the substrate into a product, the enzyme substrate complex is formed: enzyme E + substrate S enzyme substrate complex ES enzyme E + product P.
Studies on fungal polysaccharide hydrolyzing enzymes have shown a struc-tural design composed of two functional domains, a catalytic core responsible for the actual hydrolysis and a conserved cellulose-binding terminus, with an intervening, glycosylated hinge region.
A large number of genes encoding cel-lulases, hemicellulases, glucanases, amylolytic enzymes, and those hydrolyzing various oligosaccharides have been cloned from fungi. The best-studied or-ganisms are Trichoderma reesei, Phanerochaete chrysosporium, and Agaricus bisporus in respect of cellulases and hemicellulases, and several Aspergillus species in respect of amylolytic enzymes, pectinases and hemicellulases (re-views by Penttila and Saloheimo 1999; Kenealy and Jeffries 2003).
For example, papain cleavage of cellobiohydrolase (CHB) from P. chrysosporiurn separated the catalytic domain from the hinge and binding domains. Restriction mapping and sequence analysis of cosmid clones showed a cluster of three structural related CHB genes. Within a conserved region, the deduced amino acid se-quences of P. chrysosporium cbhl-1 and cbhl-2 were, respectively, 80 and 69% homologous to that of the Trichoderma reesei CBH I gene. Transcript levels of the three P. chrysosporium CHB genes varied, depending on culture conditions (review by Highley and Dashek 1998). Binding domains specific for xylan have also been identified (review by Kenealy and Jeffries 2003). Because of their valuable protein character, constitutive enzymes always present in the cell are the exception. Usually, the biosynthesis of the inducible enzymes is induced, if its presence is necessary, by the substrate or other molecules. Some work was done with regard to the regulation of extracel-lularly acting enzymes in fungi. For example with white-rot fungi, cellulase synthesis is induced in vitro by cellulose and repressed by glucose. As the wood cell-wall macromolecules are degraded outside the hypha, the most generally accepted view of the induction process is that the fungi produce a basic level of constitutive amount of enzyme that produces soluble degradation products that function as inducers.
In Phanerochaete chrysosporium, which has served as a model organism for white-rot degradation studies, cellobiose concen-tration, a product of cellulase action, is controlled in at least four ways, by f3-glucosidase, transglucosylation reactions, and two oxidative enzymes. As with cellulases, simple sugars repressed the production of most hemicellulose-degrading enzymes by white-rot fungi (review by Highley and Dashek 1998).
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