Oxalic acid quantification, oxaloacetase assay and ESI localization of P, C, and Fe from the brown rot fungus Postia placenta
C R Jordan, W V Dashek, T L Highley
The mechanism by which brown-rot fungi initiate depolymerization of holocellulose in wood remains unknown. Recently, oxalic acid (OA) has received considerable attention in cellulose breakdown by brown-rot fungi. The OA could serve as a proton donor for hydrolytic or an electron donor for oxidative (Fenton's reaction-H2O2/Fe2+) cleavages of cellulose. The acid may originate via oxaloacetase's action upon oxaloacetate. We report electron microscopic imaging (ESI) to localize Fe and HPLC/oxalic kit colorimetry to purify/quantify OA from hyphae upon agar, southern pine wood blocks (WBs) or in liquid culture. Comparative ESI at 25, 59, 110, 222, and 710 ev of hyphae grown upon agar or WBs demonstrated hyphal Fe (710 ev). Although Fe was not visualized in cell walls of uninoculated WBs, it was in certain wood cell walls of inoculated WBs. The Fe distribution differed from C and P. Oxaloacetase activity was not observed in either Amicon YM10 filter-retained intra-or extracellular fractions of liquid cultured hyphae or in homogenates from decayed WBs. In contrast, HPLC detected OA in both Postia placenta liquid cultures and decayed WBs. The less sensitive oxalic kit (mg vs. ug) did not detect OA in liquid cultures. These results constitute additional evidence for an OA Fe2+/H2O2-Fenton's mechanism for brown rot-induced cellulose degradation. However, OA's origin was not established.
Keywords: BROWN ROT; DECAY; ESI IMAGING; IRON DISTRIBUTION; OXALOACETASE; OXALIC ACID; FENTON'S REACTION
Conference: 94-05-29...06-03, Nusa Dua, Bali, Indonesia