New polymeric model substrates for the study of microbial ligninolysis

Abstract

Lignin model dimers are valuable tools for the elucidation of microbial ligninolytic mechanisms, but their low molecular weight (MW) makes them susceptible to nonligninolytic intracellular metabolism. To address this problem, we prepared lignin models in which unlabeled and ␣-14 C-labeled ␤-O-4-linked dimers were covalently attached to 8,000-MW polyethylene glycol (PEG) or to 45,000-MW polystyrene (PS). The watersoluble PEG-linked model was mineralized extensively in liquid medium and in solid wood cultures by the white rot fungus Phanerochaete chrysosporium, whereas the water-insoluble PS-linked model was not. Gel permeation chromatography showed that P. chrysosporium degraded the PEG-linked model by cleaving its lignin dimer substructure rather than its PEG moiety. C ␣-C ␤ cleavage was the major fate of the PEG-linked model after incubation with P. chrysosporium in vivo and also after oxidation with P. chrysosporium lignin peroxidase in vitro. The brown rot fungus Gloeophyllum trabeum, which unlike P. chrysosporium lacks a vigorous extracellular ligninolytic system, was unable to degrade the PEG-linked model efficiently. These results show that PEG-linked lignin models are a marked improvement over the low-MW models that have been used in the past.

Manganese-Dependent Cleavage of Nonphenolic Lignin Structures by Ceriporiopsis subvermispora in the Absence of Lignin Peroxidase

“…PEG-linked lignin model compound I ( Fig. 1A) was synthesized with a 14 C label at C ␣ as described previously (13). The model contained approximately 0.9 ␤-O-4 dimer per 8,000-average-MW PEG chain.…”

“…Ligninolytic activity of C. subvermispora in wood block cultures. Birch blocks (approximately 3 cm 3 , six replicates per experimental condition) were infused with 14 C-labeled model I (1.0 ϫ 10 5 to 1.1 ϫ 10 5 dpm per block) as described previously (13). When isotope trapping experiments were done, unlabeled compound IV (5 mg) was infused into each block with the labeled model I.…”

“…The blocks were then inoculated and adjusted to 80 to 85% water content by infusing them with a blended suspension of C. subvermispora mycelium in 0.36% (wt/vol) potato dextrose broth. The inoculated blocks were placed on Teflon spacers over pregrown C. subvermispora cultures on potato dextrose agar in 125-ml Erlenmeyer flasks (13). The flasks were fitted with gassing manifolds, incubated at 30ЊC, and flushed daily with sterile, moist air to vent evolved 14 CO 2 , which was trapped in an ethanolamine-containing cocktail for quantitation by scintillation counting (17).…”

“…To address this question, we have found conditions under which C. subvermispora degrades high-molecular-weight (MW) nonphenolic lignin structures efficiently in a defined liquid culture medium that can be assayed easily for LiP activity. By using this culture system, we have identified some of the major cleavage metabolites that C. subvermispora generates when it degrades a high-MW polyethylene glycol (PEG)-linked lignin model compound that represents the major nonphenolic ␤-O-4 structure of lignin (13). The results show that C. subvermispora cultures which express no detectable LiP activity are nevertheless able to cleave benzylic fragments from macromolecular ␤-O-4 lignin structures as LiP-producing fungi do.…”

Estudo da produção de enzimas ligninolíticas por Ceriporiopsis subvermispora

“…As isoenzimas de LiP são glicoproteínas globulares com massa molecular (M R ) em torno de 38-42 kDa, com ponto isoelétrico (pI) de 3,2-4,0 e possuem pH ótimo próximo de (CARVALHO, 2009;HOFRICHTER, 2002;KAWAI et al, 1995;AKALESWARAN;GOLD, 1988 (HOFRICHTER, 2002;JENSEN et al,1996;KAPICH;JENSEN;HAMMEL, 1999).…”