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, 65 (5), 1864-70

Production of Manganese Peroxidase and Organic Acids and Mineralization of 14C-labelled Lignin (14C-DHP) During Solid-State Fermentation of Wheat Straw With the White Rot Fungus Nematoloma Frowardii

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Production of Manganese Peroxidase and Organic Acids and Mineralization of 14C-labelled Lignin (14C-DHP) During Solid-State Fermentation of Wheat Straw With the White Rot Fungus Nematoloma Frowardii

M Hofrichter et al. Appl Environ Microbiol.

Abstract

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).

Figures

FIG. 1
FIG. 1
Activities of MnP (●) and laccase (▾) during SSF of wheat straw with N. frowardii. Enzyme activities are expressed in relation to the water content of the straw (in units per liter). Each data point is the mean enzyme activity value for three extracted flasks; the bars indicate standard deviations.
FIG. 2
FIG. 2
Separation of extracellular proteins from 23-day-old cultures of N. frowardii grown on wheat straw by anion-exchange chromatography on Sepharose-Q. (A) —, absorbance at 280 nm (OD [280 nm]); - - - , absorbance at 409 nm (OD [409 nm]); …, salt gradient (0.05 to 0.30 M NaCl). (B) Enzyme activities. Lacc, laccase.
FIG. 3
FIG. 3
Purification of pooled ligninolytic activities on a Mono-Q Sepharose column. (A) —, absorbance at 409 nm (OD [409 nm]); - - - , salt gradient. (B) Enzyme activities. Lacc, laccase.
FIG. 4
FIG. 4
IEF analysis of MnP separated from N. frowardii during SSF of wheat straw (lanes 1 and 2) and IEF analysis of MnP2 (lane 3) and MnP1 (lane 4) obtained from liquid cultures (51). MnP activity was stained with phenol red (lane 1) or ABTS (lanes 2 through 4) in the presence of Mn(II) and H2O2.
FIG. 5
FIG. 5
Accumulation of malate (⧫), fumarate (▾), and oxalate (●) during SSF of wheat straw with N. frowardii. Millimolar concentrations are expressed in relation to the water content of straw. Each data point is the mean organic acid level for three extracted culture flasks; the bars indicate standard deviations.
FIG. 6
FIG. 6
Mineralization of 14C-ring-labelled synthetic lignin (22,000 dpm) by N. frowardii during growth on wheat straw (●). ⧫, noninoculated control. The bars indicate standard deviations (n = 3).
FIG. 7
FIG. 7
Production of 14CO2 from 14C-DHP by purified N. frowardii MnP in a cell-free system. Each reaction mixture contained 2 U of MnP, 30 mM malate, 5 mM fumarate, 1 mM MnCl2, 100 mg of unlabelled DHP per liter, and 22,000 dpm of 14C-DHP and was incubated on a rotary shaker at 37°C in the dark. The data points are means of three replicates, and the bars indicate standard deviations. Symbols: ■, complete reaction mixture; ●, reaction mixture without fumarate; ⧫, control without MnP.

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