|Title||Physiological roles and metabolism of fungal aryl alcohols|
|Author(s)||Jong, E. de|
|Source||Agricultural University. Promotor(en): J.A.M. de Bont, co-promotor(en): M.M.G.R. Bol; J.A. Field. - S.l. : De Jong - ISBN 9789054851943 - 224|
|Publication type||Dissertation, internally prepared|
|Keyword(s)||enzymen - bosbouw - pulpbereiding - biodegradatie - oxidoreductasen - peroxidasen - katalase - basidiomycotina - scheurvorming - decompositie - degradatie - lignine - chemische verbindingen - enzymes - forestry - pulping - biodegradation - oxidoreductases - peroxidases - catalase - basidiomycotina - cracking - decomposition - degradation - lignin - chemical compounds|
The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism Phanerochaete chrysosporium and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.
Aryl alcohols In the physiology of white-rot fungi. White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H 2 O 2 .
The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H 2 O 2 either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in P. chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium .
One of the best Poly R decolorizing strains, Bjerkandera sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of Bjerkandera sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H 2 O 2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H 2 O 2 . Bjerkandera sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of de novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the denovo biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H 2 O 2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.
It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus Bjerkandera sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg -1wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.
Degradation of aryl alcohols by fungi. In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). Penicilliumsimplicissimum was selected for the characterization of the veratryl alcohol degradation route. P. simplicissimum oxidized veratryl alcohol via a NAD(P) +-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) +-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent O -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of P. simplicissimum is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N 3-histidyl)FAD from pH dependent fluorescence quenching (p Ka = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol.