The involvement of hydroxyl-radical-producing glycoprotein from the white-rot basidiomycete Ceriporiopsis subvermispora in wood decay

IRG/WP 09-10688

H Tanaka, Y Inoue, T Morikawa, S Itakura, A Enoki

White-rot decay can be divided into two subtypes. One type involves the simultaneous degradation of all wood components through the formation of erosion troughs with a progressive thinning of wood cell walls. This type of decay is consistent with a model in which several polymer-degrading enzymes act on the exposed surfaces of the wood cell walls, producing progressive erosion from the lumen to the middle lamella. The second type of white rot is selective lignin degradation, carried out by a relatively small number of fungi. In this case, lignin and non-cellulosic polysaccharide are removed without progressive thinning of the wood cell walls. Ceriporiopsis subvermispora is one of the best examples of a selective lignin degrader. Although the reason why this fungus selectively degrades lignin is still unclear, the lack of a complete cellulolytic enzymatic complex has been considered as one of the motives for inexpressive cellulose degradation. Moreover, it has been reported that this fungus secretes a number of low-molecular-mass compounds including oxalic acid during solid-state fermentation of wheat straw [1] and several fatty acids during solid-state fermentation of wood [2,3]. Independent of their origins, low-molecular-mass compounds may play key roles in the overall wood biodegradation process. During wood decay, the white-rot basidiomycete Phanerochaete chrysosporium secretes low-molecular-mass glycoproteins that catalyze a redox reaction between O2 and electron donors to produce hydroxyl radical (•OH). This reaction accounts for most of the •OH produced in wood-degrading cultures of P. chrysosporium. In combination with phenol oxidases, •OH is believed to play a role in lignin degradation [4,5]. In our previous study [6], cellulose and lignin depolymerization, as well as the production of the hydrolytic enzymes, carboxymethylcellulase (CMCase) and Avicelase (exo-1,4-β-D-cellobiohydrolase), and the ligninolytic enzymes, laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP), have been determined during the wood-decay process of C. subvermispora. During the 12-week incubation with Japanese beech wood, C. subvermispora continuously produced at least one of three phenol oxidases: laccase was produced initially, followed by Mn-independent peroxidase activity peaking at 6 weeks and MnP activity peaking at 10 weeks. Lignin peroxidase and CMCase activities peaked after 3 weeks of incubation. Avicelase activity was present throughout the incubation period, although the activity was very low. Furthermore, it was also shown that the low-molecular-mass fraction of the extracellular medium, which catalyzes a redox reaction between O2 and electron donors to produce •OH, may act synergistically with the enzymes to degrade wood cell walls. A. low molecular-weight, extracellular substance with one-electron oxidizing activity was isolated from C. subvermispora. The substance was partially purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration, and DEAE-Sepharose ion-exchange chromatography. The partially purified material was a glycoprotein composed of 32% protein and 46 % neutral carbohydrate, containing 0.05% Fe(II) by weight. Tricine-SDS-PAGE showed 2 bands with a molecular mass of around 13000. One mg of the partially purified glycoprotein reduced 1.3 μmol of Fe(III) to Fe(II) and contained at least 0.4 μmol of α-hydroxyketone or endiol groups. Most of the α-hydroxyketone groups were 1-amino-2-ketose produced by the condensation of side-chain amino groups and carbohydrates. We have reported that the white-rot fungi Irpex lacteus [7], P. chrysosporium [4,5], and Trametes versicolor [8] produce at least one phenol-oxidizing enzyme, as well as •OH produced by agents other than phenol oxidase. Herein we demonstrate that C. subvermispora produces laccase, MnP, LiP, and a low-molecular-mass fraction which can generate ethylene from 2-keto-4-thiomethylbutyric acid due to the oxidizing activity of •OH. This low-molecular-mass fraction described above has very similar properties to the •OH-producing glycoprotein from the other white-rot fungi [4,5,8]. These glycoproteins catalyze a redox reaction between O2 and electron donors to produce •OH. The Fenton system [Fe(II) and H2O2], which is known to depolymerize cellulose [9], can be generated in white-rot-fungal cultures, since H2O2 is present and Fe(II) can be formed by the activity of enzymes, such as cellobiose dehydrogenase [10] or MnP [11], and also by the Fe(III)-reducing activity of fungal-produced hydroxyaromatic carboxylic acids [12]. The glycoprotein reduced O2 to H2O2 and Fe(III) to Fe(II), and thus could generate •OH via a Fenton reaction. Thus the •OH-producing glycoprotein found in white-rot fungi [4,5,8] could also be involved. We propose that during wood degradation by C. subvermispora, laccase and MnP preferentially degrade lignin, in concert with a system that produces •OH. In addition, this fungus’s incomplete cellulase system and its •OH-generating system may act synergistically to degrade and metabolize cellulose.

Keywords: wood degradation, Ceriporiopsis subvermispora, White-rot fungi, Hydroxyl-radical-producing glycoprotein

Conference: 09-05-24/28 Beijing, China

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