The ability of the white rot fungus Ceriporiopsis subvermispora to mineralize 14C-synthetic lignin was studied under different culture conditions, and the levels of two extracellular enzymes were monitored. The highest mineralization rates (28% after 28 days) were obtained in cultures containing a growth-limiting amount of nitrogen source (1.0 mM ammonium tartrate); under this condition, the levels of manganese peroxidase (MnP) and laccase present in the culture supernatant solutions were very low compared with cultures containing 10 mM of the nitrogen source. In contrast, cultures containing a limiting concentration of the carbon source (0.1% glucose) showed low levels of both enzymes and also very low mineralization rates compared with cultures containing 1% glucose. Cultures containing 11 ppm of Mn(II) showed a higher rate of mineralization than those containing 0.3 or 40 ppm of this cation. Levels of MnP and laccase were higher when 40 ppm of Mn(II) was used. Mineralization rates were slightly higher in cultures flushed daily with oxygen, whereas laccase levels were lower and MnP levels were approximately the same as in cultures maintained under an air atmosphere. The presence of 0.4 mM veratryl alcohol reduced both mineralization rates and MnP levels, without affecting laccase levels. Lignin peroxidase activity was not detected under any condition. Addition of purified lignin peroxidase to the cultures in the presence or absence of veratryl alcohol did not enhance mineralization rates significantly. White rot fungi degrade lignin by the action of enzymes that are secreted into the medium. Two extracellular peroxidases, lignin peroxidase (LiP) (15, 30) and manganese peroxidase (MnP) (14, 25), and a copper-containing phenol oxidase (laccase) (6) have been widely studied, and evidence that they participate in lignin depolymerization has accumulated. Laccase and MnP are probably more widely distributed among white rot fungi than is LiP. Phanerochaete
The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene.
The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the ironmolybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe 7-8 S x precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe 3 S 4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.
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