6 Wood Discoloration

90 4 Wood Cell Wall Degradation
cellular enzymes (ectoenzymes) into smaller fragments, which are taken up
and then metabolized by intracellular enzymes to energy and fungal biomass.
Independent of this place of action, an exoenzyme attacks at the end of a macromolecular
substrate, while an endoenzyme splits within the molecule. These
four terms are sometimes mixed up.
Occurrence and distribution of enzymes and metabolites inside hyphae, in
the hyphal slime layer, and within the attacked woody tissue were investigated
by means of immunological methods and electron microscopy (Sprey 1988;
Goodell et al. 1988; Srebotnik et al. 1988a; Blanchette et al. 1989, 1990; Daniel
et al. 1989, 1990; Srebotnik and Messner 1990; Kim 1991; Green et al. 1991b;
Kim et al. 1991a, 1991b, 1992, 1993; Lackner et al. 1991; Blanchette and Abad
1992). TEM of immunogold-labeled hyphae of Trametes versicolor grown on
carboxymethylcellulose localized β-glucosidase on the plasmalemma, in the
hyphal cell wall, and in the hyphal sheath (review by Highley and Dashek 1998).
Simple methods are used in screening tests to detect enzymes and to determine
their activity. For example, a cellulose is added to a fungal culture, whose
cellulolytic enzymes produce glucose. The glucose of the sampled culture filtrate
reduces a test compound, which is added in oxidized form and changes
its color by reduction. At a specific wavelength, the quantity of the converted
test substance and thus of the developed glucose is measured and the activity
of “cellulolytic enzymes” is calculated. Remazol brilliant blue, which binds
to cellulose and hemicellulose by a microbially relatively inert linkage, may
be mixed in agar. If cellulolytic or hemicellulolytic microorganisms or their
enzymes are present, the still colored degradation products are released and
clearing zones occur around the active colony, which can be also quantified
(Schmidt and Kebernik 1988; Takahashi et al. 1992). For detailed investigations,
various purification and enrichment steps may be used (chromatography, electrophoresis,
etc.).
The current unit of enzyme activity is katal (catalytic activity, kat), although
in practice the old definition U is still used (Table 4.1).
Microbial wood degradation is influenced by several major characteristics
of the substrate wood (Table 4.2).
Accessory compounds in the heartwood as well as resin excretion and wound
reactions after wounding inhibit the colonization and spread of microorganisms
within the tree (Chap. 3.1).
The polymeric structure of the nutrients cellulose, hemicelluloses, and
lignin requires that the degrading agents act outside the hypha. Cowling (1961)
first stated that the known enzymes of the time were too large to penetrate
intotheinteriorofthewoodcellwallandhypothesizedapossibleexistence
of a small mass enzyme. The molecular weights of cellulases range from 13–
61 kDa (Fengel and Wegener 1989). A cellulase of 40 kDa can exhibit a thickness
of about 4 nm and a length of 18 nm (Messner and Srebotnik 1989). Frequently,
about an 8 nm size was measured (Reese 1977; Messner and Stachelberger
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