12.2.1 Lignin Degradation
Lignin is formed by random cross-linking of three monomer phenylpropanoid units: p-hydroxycinnamyl (coumaryl) alcohol, 4-hydroxy-3-methoxycinnamyl (coniferyl) alcohol and 3,5-dimethoxy-4-hydroxycinnamyl (sinapyl) alcohol with several different carbon-carbon and carbon-oxygen linkages (Figure 12.2). Lignin encrusts the cellulose microfibrils within plant cell walls, giving the vascular plant the rigidity and protecting the plant from weather, insects and pathogenic organisms. It is remarkable, therefore, that a few fungi belonging to Basidiomycotina, such as Trametes versicolor, Bjerkandera adusta, Hypholoma fasciculare, Stereum hirsutum, Gymnopilus penetrans, Agaricus bisporus, Pleurotus ostratus and Lenti-mus edodes (http://www.ftns.wau.nl/imb/research/wrf.htnl), can selectively degrade lignin and access the carbohydrate polymers within the cell wall.
The screening of active ligninolytic fungi is done by inoculating wood blocks with the fungus and estimating lignin loss by chemical analyses and transmission electron microscopy. Lignin-degrading activity is measured by measuring the evolution of 14CO2 from 14C-labeled synthetic lignin prepared by polymerizing 14C-labelled p-hydroxycin-namyl alcohols with horseradish peroxidase, or by the oxidation of a lignin model compound, veratryl (3,4-dimethoxybenzyl) alcohol to veratraldehyde in the presence of H2O2. Electron microscopy of wood decayed by white-rot fungi revealed that lignin is degraded at some distance from the hyphae, suggesting that the hyphae produce a highly reactive oxygen species that diffuses out and depolymerizes lignin by breaking the carbon-oxygen and the carbon-carbon bonds. The inability of large-size enzymes to diffuse into wood suggests that fungi employ smaller reactive oxygen species that cleaves C-C bonds. An extracellularly produced lignin-degrading enzyme resembling peroxidase (haem protein) in spectral properties was isolated from Phanerochaete chrysosporium (Basidiomycotina). Spectacular photographs of this fungus may be viewed online (http://botit.botany.wisc.edu/tomes_fungi/may97.html). The fungus grows optimally at about 40°C and has been isolated from stored wood chips used for the manufacture of paper and in saw-mill waste that attains high temperatures. The extracellular enzyme
catalyzed C-C cleavage in lignin model compounds (Tien and Kirk, 1984) and required hydrogen peroxide for activity. Peroxidases catalyze reactions wherein hydrogen peroxide is reduced, while a substrate is oxidized simultaneously,
where AH2 is a reduced substrate and A is the oxidized substrate. Hydrogen peroxide is a powerful oxidant and may be produced by oxidases that oxidize sugars to sugar lactones (Hammel, 1997). Immunochemical localization using gold-labeled antiserum in sections of white-rotted wood shows that lignin peroxidase is present in cell walls undergoing delignification. Two types of ligninase have been found: those which require manganese for catalytic activity (MnP) and those that do not require a metal ion for activity (LiP). The enzymes can oxidize aromatic compounds containing free phenolic groups by removal of one electron from the aryl rings to form an aromatic cation radical. (A radical is a molecular fragment having one or more unpaired electrons. It pairs up with other electrons to make new chemical bonds, making radicals highly reactive). The radicals break down the lignin polymer and explain how lignin is degraded at sites some distance away from the hyphae. In white rot fungi, the extracellular H2O2 required for the activity of lignin peroxidases is produced from the oxidation of glyoxal and methylglyoxal—metabolites
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