Specific objectives: 1) To test the hypothesis that aDal (lanardfie) Salubilizatibn and the subse... more Specific objectives: 1) To test the hypothesis that aDal (lanardfie) Salubilizatibn and the subsequent depdymerization of the dubilized coal macromolecules are distinct events in linin degrading fungi. tn addition to T. vemMur, phanerochaete chrysospo&m, anather lignin degrading hrngus that also has the ability to solubilize coal, will be studied. 2) To test the hypothesis that the proaesses d mal (leonardite) dubilizatbn and mal mammalewie depolymerization in lignin degrading fungi can be regulated by altering the nutritional status of the microorganism. Coal mlubilization is expected to occur in notn'ent rich media whereas depolymerization of solubitized coal macromolecules is expected to OCoLir in nutrient limited media. 3) To determine the role of extracellular eruymes {lactases, lignin peroxidases and Mn peroxidases) that qye secreted by lignin degrading fungi during cod sdubiliation w coal macromoleatle depolymetitation.
Specific objectives: 1) To test the hypothesis that coal (leonardite) Solubilization and the subs... more Specific objectives: 1) To test the hypothesis that coal (leonardite) Solubilization and the subsequent depolymerization of the solubilized coal macromolecules are distinct events in lignin degrading fungi. In addition to T. versico/or, Phanerochaete chrysosporium, another lignin degrading fungus that also has the ability to solubilize coal, will be studied. 2) To test the hypothesis that the processes of coal (leonardite) solubilization and coal macro molecule depolymerization in lignin degrading fungi can be regulated by altering the nutritional status of the microorganism. Coal solubilization is expected to occur in nutrient rich media whereas depolymerization of solubilized coal macromolecules is expected to occur in nutrient limited media. 3) To determine the role of extracellular enzymes (Iaccases, lignin peroxidases and Mn peroxidases) that are secreted by lignin degrading fungi during coal solubilization or coal macro molecule depolymerization. 4) To assess the role of enzymatically generated oxygen radicals, non-radical active oxygen species, veratryl alcohol radicals and Mn+++ complexes in coal macro molecule depolymerization. 5) To characterize products of coal solubilization and coal macro molecule depolymerization that are formed by T. versicdor and P, chrysosporium and their respective extracellular enzymes. Solubilization products formed using oxalic acid and other metal chelators will also be characterized and compared. Overview During this reporting period our efforts focused on: 1) Determining the effect of pH on coal solubilization by oxalate ion and other biologically important compounds that might function as metal chelators; 2) The role of laccase in low rank coal solubilization and metabolism; 3) Decolorization of soluble coal macromolecule by P. chrysosporium and T. versicolor in solid agar media and 4) Solubilization of low rank coal in slurry cultures and solid phase reactors containing P. chrysosporium. Methods and materials Several of the methods used have been described in previously submitted quarterly DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or uscfulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or senrice by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, tccommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
Purified cytochrome P-450,,, from bovine adrenal cortex mitochondria after treatment with BrCN yi... more Purified cytochrome P-450,,, from bovine adrenal cortex mitochondria after treatment with BrCN yielded a core peptide which retains heme. The amino acid composition of this peptide was similar to that of the analogous peptide isolated from cytochrome P-450,of Pseudomonas putida. Adrenodoxin and cholesterol association with P-450,, was analyzed in nonionic Tween 20 micelles where cholesterol appears to be fully in equilibrium with the cytochrome. Adrenodoxin binding to cholesterol-free P-450.,, was observed by a type I spectral shift in the cytochrome (Kd = 4 X lo" M). Binding to the cholesterol-P-450,, complex was over 10 times stronger (Kd = 3 X M). Binding of adrenodoxin to both free enzyme and cholesterol complex was unaffected by Tween 20, indicating a clear separation of the adrenodoxin binding site from the hydrophobic membrane binding domain. Adrenodoxin binding is driven by a large increase in entropy (AS = 30 e.u., A H = 0 kcal), while cholesterol activation of this binding is a consequence of a further increase in A S (from 30 to 53 e.u.) which more than offsets an increase in A H (5.7 kcal). The spin states of the complexes of cytochrome P-450.,, with both cholesterol and adrenodoxin and of the ternary complex were insensitive to changes of temperature (5-35"C), but the high spin content of the cholesterol complex in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer was raised by increased ionic strength (200 m~ KC1,83%) and by the binding of adrenodoxin (92%). The Kd for adrenodoxin-P-450,,, complex and the apparent K,,, for adrenodoxin in cholesterol side chain cleavage reaction both increased exponentially with ionic strength. In contrast, the analogous constants for cholesterol were insensitive to ionic strength. The initial velocity patterns with varied adrenodoxin and cholesterol intersected below the horizontal axis, the apparent K,,, for each reactant increasing with increasing concentrations of the second reactant. The true K,,, for each reactant was manyfold greater than the respective Kd for complex formation with P-450,,,.
The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes ... more The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes called lignin peroxidases which are involved in the degradation of both lignin and a number of persistent environmental pollutants. Lignin peroxidase isozyme H2, a glycosylated protein of approximately 40 kDa, contains a single heme. X-ray absorption spectroscopy (XAS) has been used to probe the local environment of the iron in the active site of resting enzyme, reduced enzyme, and compound 111. For the native and reduced forms, respectively, the average Fe-pyrrole nitrogen distances are 2.055 and 2.02 A (f0.015 A); the Fe-proximal nitrogen distance is 1.93 and 1.91 A (f0.02 A) while the Fe-distal ligand distance is 2.17 and 2.10 A (f0.03 A). Although the results are not as well-defined, the active-site structure of compound I11 is largely 2.02 f 0.015 A for the average Fe-pyrrole nitrogen distance, 1.90 f 0.02 for the Fe-proximal nitrogen, and 1.74 f 0.03 A for the Fe-distal ligand distance. The heme iron-pyrrole nitrogen distnce is more expanded in ligninase H 2 than in other peroxidases. The possible significance of this is discussed in relation to other heme proteins.
Applied Microbiology and Biotechnology, Sep 1, 1989
Extensive biodegradation of [14C]-2,4,5-trichlorophenoxyacetic acid ([14C]-2,4,5-T) by the white ... more Extensive biodegradation of [14C]-2,4,5-trichlorophenoxyacetic acid ([14C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [14C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [14C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0% + 2.0% of the [14C]-2,4,5-T initially present was degraded to 14CO2. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [14C]-2,4,5-T metabolites. It was also shown that only ~ 5% of the [14C]-2,4,5-T initially present in cultures remained as undegraded [14C]-2,4,5-T. In incubations composed of [~4C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%+ 3.6% of the [~4C]-2,4,5-T initially present was converted to ~4CO2 after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.
During this reporting period we have further studied the oxidation of soluble coal macromolecules... more During this reporting period we have further studied the oxidation of soluble coal macromolecules by lignin peroxidase from Phanerochaete chrysosporium. Previous studies by others have suggested that a soluble fraction (coal macromolecule B-III) from a nitric acid solubilized North Dakota Lignite is depolymerized by this enzyme. Our investigations indicate that fraction B-III is a substrate for lignin peroxidase as this material is decolorized in the presence of lignin peroxidase H8 and hydrogen peroxide. Of interest, however, is the observation that little, if any, depolymerization of this material occurs. Instead, it appears that lignin peroxidase and coal macromolecule B-III form a precipitate. These results are similar to those observed in our investigations of lignin peroxidase mediated oxidation of oxalate solubilize coal macromolecule. Previous studies in our laboratory using a spectrophotometric assay suggested that, in addition to oxalate, several other fungal metabolites are able to solubilize leonardite. We have reinvestigated this phenomenon using a more reliable gravimetric procedure for assessing solubilization. Our results confirm our earlier findings that malate, oxaloacetate and citrate are effective solubilizing agents whereas succinate, fumarate and α-ketoglutarate solubilize relatively small amounts of leonardite. Finally, we have studied the composition of the insoluble material remaining following extensive solubilization by sodium oxalate. The ratio of hydrogen to carbon is increased in the insoluble material relative to the parent leonardite. However, the ratio of oxygen to carbon is also increased in the insoluble material. Thus, the insoluble material does not appear to be more highly reduced that the parent leonardite and is not likely to be a better fuel that the parent material.
During this reporting period we have further studied the oxidation of soluble coal macromolecules... more During this reporting period we have further studied the oxidation of soluble coal macromolecules by lignin peroxidase from Phanerochaete chrysosporium. Previous studies by others have suggested that a soluble fraction (coal macromolecule B-III) from a nitric acid solubilized North Dakota Lignite is depolymerized by this enzyme. Our investigations indicate that fraction B-III is a substrate for lignin peroxidase as this material is decolorized in the presence of lignin peroxidase H8 and hydrogen peroxide. Of interest, however, is the observation that little, if any, depolymerization of this material occurs. Instead, it appears that lignin peroxidase and coal macromolecule B-III form a precipitate. These results are similar to those observed in our investigations of lignin peroxidase mediated oxidation of oxalate solubilize coal macromolecule. Previous studies in our laboratory using a spectrophotometric assay suggested that, in addition to oxalate, several other fungal metabolites are able to solubilize leonardite. We have reinvestigated this phenomenon using a more reliable gravimetric procedure for assessing solubilization. Our results confirm our earlier findings that malate, oxaloacetate and citrate are effective solubilizing agents whereas succinate, fumarate and α-ketoglutarate solubilize relatively small amounts of leonardite. Finally, we have studied the composition of the insoluble material remaining following extensive solubilization by sodium oxalate. The ratio of hydrogen to carbon is increased in the insoluble material relative to the parent leonardite. However, the ratio of oxygen to carbon is also increased in the insoluble material. Thus, the insoluble material does not appear to be more highly reduced that the parent leonardite and is not likely to be a better fuel that the parent material.
Portions of this document may be illegible in electronic image products. Images are produced from... more Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. Disclaimer This rep rt was prepared as an a writ of work sponsored by an agency of the United States Government.
The wood-rotting fungus Phanerochaete chrysosporium is able to degrade a wide variety of environm... more The wood-rotting fungus Phanerochaete chrysosporium is able to degrade a wide variety of environmentally-persistent organic pollutants to carbon dioxide. The unique biodegradative abilities of this fungus are due, in part, to lignin peroxidases, oxidative enzymes that are secreted in response to nutrient deprivation. Lignin peroxidases catalyze the initial oxidation of many of the organic pollutants that are degraded by this fungus. They also mediate the initial oxidation of N,N,N{prime},N{prime},N{double_prime},N{double_prime}-hexamethylpararosaniline, several azo dyes and certain polycyclic aromatic hydrocarbons. Lignin peroxidases also mediate oxidative dechlorination. For example, lignin peroxidases oxidize pentachlorophenol to 2,3,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione. Similarly, these enzymes mediate oxidative oligomerization of 4-chloroaniline, resulting in production of several dimers, trimers and tetramers and net dechlorination of the aromatic ring. Interestingly, m...
Boiling Air Force Base, D.C. 20332-6448 AvDilability Cedp l j~~tst ipecinI 92-30010 I iEIIelligYl... more Boiling Air Force Base, D.C. 20332-6448 AvDilability Cedp l j~~tst ipecinI 92-30010 I iEIIelligYli[. 9'2_. 1 2-.,5 2
The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hy... more The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distillation product obtained from coal tar) was demonstrated. Analysis by capillary gas chromatography and high-performance liquid chromatography showed that at least 22 PAHs, including all of the most abundant PAH components present in anthracene oil, underwent 70 to 100% disappearance during 27 days of incubation with nutrient nitrogen-limited cultures of this fungus. Because phenanthrene is the most abundant PAH present in anthracene oil, this PAH was selected for further study. In experiments in which [14C]phenanthrene was incubated with cultures of P. chrysosporium containing anthracene oil for 27 days, it was shown that 7.7% of the recovered radiolabeled carbon originally present in [14C]phenanthrene was metabolized to 14CO2 and 25.2% was recovered from the aqueous fraction, while 56.1 and 11.0% were recovered from the methyl...
A straightforward procedure using density functional theory (M06 2X) coupled with a group-equival... more A straightforward procedure using density functional theory (M06 2X) coupled with a group-equivalent approach is described that was used to calculate gas-phase heat of formation (Δf H°g,298) values for buckminsterfullerene (C60), C70 fullerene (C70), corannulene, coronene, and sumanene. This procedure was also used to calculate exceptionally accurate Δf H°g,298 values for a variety of single-ring aromatic and 2-7 ring polycyclic aromatic hydrocarbons (PAHs) as well as a large selection of other hydrocarbons and phenols. The approach described herein is internally consistent, and results for C60, C70, corannulene, coronene, and sumanene are in very close agreement with results reported by others who used higher-level computational theory. Statistical analysis of a test set containing benzene and 18 two to seven ring PAHs demonstrated that by using this approach a mean absolute deviation (MAD) and a root-mean-square deviation (RMSD) of 0.8 and 1.3 kJ/mol, respectively, were achieved for reference/experimental Δf H°g,298 values versus calculated/predicted Δf H°g,298 values. For statistical analysis of a larger test set containing 235 aromatic and aliphatic hydrocarbons and phenols, a MAD and a RMSD of 1.2 and 1.9 kJ/mol, respectively, were achieved for reference/experimental Δf H°g,298 values versus calculated/predicted Δf H°g,298 values.
Microsomes, Drug Oxidations and Chemical Carcinogenesis, 1980
Cytochrome P-450 and NADPH cytochrome c (P-450) reductase were purified from bovine adrenocortica... more Cytochrome P-450 and NADPH cytochrome c (P-450) reductase were purified from bovine adrenocortical (BAC) microsomes. Cytochrome P-450 AM-1 purified to 10–12 nanomoles P-450/mg protein by AM 2 SO 4 fractionation and hydrophobic interaction chromatography showed essentially one band by SDS-page (>95%) with a MW = 52,000. Fraction P-450 AM-2 with a specific content of 3-4 nanomoles P-450/mg protein contained two hemeproteins with M.W. of 49,000 and 52,000. Partially purified reductase reduced cytochrome P-450. Adrenodoxin was not required nor did it stimulate reduction. Photoaffinity labeling studies show the inhibitor derived label 1-(4-azidophenyl)imidazole to have high affinity for BAC microsomal P-450. Labeling experiments using partially purified BAC microsomal P-450 afforded stoichiometric label incorporation.
Al (noting that the initial death toll was reported by state-owned television as 3,934, at the sa... more Al (noting that the initial death toll was reported by state-owned television as 3,934, at the same time that some government media was indicating it could be 10,000 and the Thai Foreign Minister indicated it could be 15,000 after meeting with Myanmar's ambassador).
Specific objectives: 1) To test the hypothesis that aDal (lanardfie) Salubilizatibn and the subse... more Specific objectives: 1) To test the hypothesis that aDal (lanardfie) Salubilizatibn and the subsequent depdymerization of the dubilized coal macromolecules are distinct events in linin degrading fungi. tn addition to T. vemMur, phanerochaete chrysospo&m, anather lignin degrading hrngus that also has the ability to solubilize coal, will be studied. 2) To test the hypothesis that the proaesses d mal (leonardite) dubilizatbn and mal mammalewie depolymerization in lignin degrading fungi can be regulated by altering the nutritional status of the microorganism. Coal mlubilization is expected to occur in notn'ent rich media whereas depolymerization of solubitized coal macromolecules is expected to OCoLir in nutrient limited media. 3) To determine the role of extracellular eruymes {lactases, lignin peroxidases and Mn peroxidases) that qye secreted by lignin degrading fungi during cod sdubiliation w coal macromoleatle depolymetitation.
Specific objectives: 1) To test the hypothesis that coal (leonardite) Solubilization and the subs... more Specific objectives: 1) To test the hypothesis that coal (leonardite) Solubilization and the subsequent depolymerization of the solubilized coal macromolecules are distinct events in lignin degrading fungi. In addition to T. versico/or, Phanerochaete chrysosporium, another lignin degrading fungus that also has the ability to solubilize coal, will be studied. 2) To test the hypothesis that the processes of coal (leonardite) solubilization and coal macro molecule depolymerization in lignin degrading fungi can be regulated by altering the nutritional status of the microorganism. Coal solubilization is expected to occur in nutrient rich media whereas depolymerization of solubilized coal macromolecules is expected to occur in nutrient limited media. 3) To determine the role of extracellular enzymes (Iaccases, lignin peroxidases and Mn peroxidases) that are secreted by lignin degrading fungi during coal solubilization or coal macro molecule depolymerization. 4) To assess the role of enzymatically generated oxygen radicals, non-radical active oxygen species, veratryl alcohol radicals and Mn+++ complexes in coal macro molecule depolymerization. 5) To characterize products of coal solubilization and coal macro molecule depolymerization that are formed by T. versicdor and P, chrysosporium and their respective extracellular enzymes. Solubilization products formed using oxalic acid and other metal chelators will also be characterized and compared. Overview During this reporting period our efforts focused on: 1) Determining the effect of pH on coal solubilization by oxalate ion and other biologically important compounds that might function as metal chelators; 2) The role of laccase in low rank coal solubilization and metabolism; 3) Decolorization of soluble coal macromolecule by P. chrysosporium and T. versicolor in solid agar media and 4) Solubilization of low rank coal in slurry cultures and solid phase reactors containing P. chrysosporium. Methods and materials Several of the methods used have been described in previously submitted quarterly DISCLAIMER Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or uscfulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or senrice by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, tccommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.
Purified cytochrome P-450,,, from bovine adrenal cortex mitochondria after treatment with BrCN yi... more Purified cytochrome P-450,,, from bovine adrenal cortex mitochondria after treatment with BrCN yielded a core peptide which retains heme. The amino acid composition of this peptide was similar to that of the analogous peptide isolated from cytochrome P-450,of Pseudomonas putida. Adrenodoxin and cholesterol association with P-450,, was analyzed in nonionic Tween 20 micelles where cholesterol appears to be fully in equilibrium with the cytochrome. Adrenodoxin binding to cholesterol-free P-450.,, was observed by a type I spectral shift in the cytochrome (Kd = 4 X lo" M). Binding to the cholesterol-P-450,, complex was over 10 times stronger (Kd = 3 X M). Binding of adrenodoxin to both free enzyme and cholesterol complex was unaffected by Tween 20, indicating a clear separation of the adrenodoxin binding site from the hydrophobic membrane binding domain. Adrenodoxin binding is driven by a large increase in entropy (AS = 30 e.u., A H = 0 kcal), while cholesterol activation of this binding is a consequence of a further increase in A S (from 30 to 53 e.u.) which more than offsets an increase in A H (5.7 kcal). The spin states of the complexes of cytochrome P-450.,, with both cholesterol and adrenodoxin and of the ternary complex were insensitive to changes of temperature (5-35"C), but the high spin content of the cholesterol complex in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer was raised by increased ionic strength (200 m~ KC1,83%) and by the binding of adrenodoxin (92%). The Kd for adrenodoxin-P-450,,, complex and the apparent K,,, for adrenodoxin in cholesterol side chain cleavage reaction both increased exponentially with ionic strength. In contrast, the analogous constants for cholesterol were insensitive to ionic strength. The initial velocity patterns with varied adrenodoxin and cholesterol intersected below the horizontal axis, the apparent K,,, for each reactant increasing with increasing concentrations of the second reactant. The true K,,, for each reactant was manyfold greater than the respective Kd for complex formation with P-450,,,.
The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes ... more The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes called lignin peroxidases which are involved in the degradation of both lignin and a number of persistent environmental pollutants. Lignin peroxidase isozyme H2, a glycosylated protein of approximately 40 kDa, contains a single heme. X-ray absorption spectroscopy (XAS) has been used to probe the local environment of the iron in the active site of resting enzyme, reduced enzyme, and compound 111. For the native and reduced forms, respectively, the average Fe-pyrrole nitrogen distances are 2.055 and 2.02 A (f0.015 A); the Fe-proximal nitrogen distance is 1.93 and 1.91 A (f0.02 A) while the Fe-distal ligand distance is 2.17 and 2.10 A (f0.03 A). Although the results are not as well-defined, the active-site structure of compound I11 is largely 2.02 f 0.015 A for the average Fe-pyrrole nitrogen distance, 1.90 f 0.02 for the Fe-proximal nitrogen, and 1.74 f 0.03 A for the Fe-distal ligand distance. The heme iron-pyrrole nitrogen distnce is more expanded in ligninase H 2 than in other peroxidases. The possible significance of this is discussed in relation to other heme proteins.
Applied Microbiology and Biotechnology, Sep 1, 1989
Extensive biodegradation of [14C]-2,4,5-trichlorophenoxyacetic acid ([14C]-2,4,5-T) by the white ... more Extensive biodegradation of [14C]-2,4,5-trichlorophenoxyacetic acid ([14C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [14C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [14C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0% + 2.0% of the [14C]-2,4,5-T initially present was degraded to 14CO2. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [14C]-2,4,5-T metabolites. It was also shown that only ~ 5% of the [14C]-2,4,5-T initially present in cultures remained as undegraded [14C]-2,4,5-T. In incubations composed of [~4C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%+ 3.6% of the [~4C]-2,4,5-T initially present was converted to ~4CO2 after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.
During this reporting period we have further studied the oxidation of soluble coal macromolecules... more During this reporting period we have further studied the oxidation of soluble coal macromolecules by lignin peroxidase from Phanerochaete chrysosporium. Previous studies by others have suggested that a soluble fraction (coal macromolecule B-III) from a nitric acid solubilized North Dakota Lignite is depolymerized by this enzyme. Our investigations indicate that fraction B-III is a substrate for lignin peroxidase as this material is decolorized in the presence of lignin peroxidase H8 and hydrogen peroxide. Of interest, however, is the observation that little, if any, depolymerization of this material occurs. Instead, it appears that lignin peroxidase and coal macromolecule B-III form a precipitate. These results are similar to those observed in our investigations of lignin peroxidase mediated oxidation of oxalate solubilize coal macromolecule. Previous studies in our laboratory using a spectrophotometric assay suggested that, in addition to oxalate, several other fungal metabolites are able to solubilize leonardite. We have reinvestigated this phenomenon using a more reliable gravimetric procedure for assessing solubilization. Our results confirm our earlier findings that malate, oxaloacetate and citrate are effective solubilizing agents whereas succinate, fumarate and α-ketoglutarate solubilize relatively small amounts of leonardite. Finally, we have studied the composition of the insoluble material remaining following extensive solubilization by sodium oxalate. The ratio of hydrogen to carbon is increased in the insoluble material relative to the parent leonardite. However, the ratio of oxygen to carbon is also increased in the insoluble material. Thus, the insoluble material does not appear to be more highly reduced that the parent leonardite and is not likely to be a better fuel that the parent material.
During this reporting period we have further studied the oxidation of soluble coal macromolecules... more During this reporting period we have further studied the oxidation of soluble coal macromolecules by lignin peroxidase from Phanerochaete chrysosporium. Previous studies by others have suggested that a soluble fraction (coal macromolecule B-III) from a nitric acid solubilized North Dakota Lignite is depolymerized by this enzyme. Our investigations indicate that fraction B-III is a substrate for lignin peroxidase as this material is decolorized in the presence of lignin peroxidase H8 and hydrogen peroxide. Of interest, however, is the observation that little, if any, depolymerization of this material occurs. Instead, it appears that lignin peroxidase and coal macromolecule B-III form a precipitate. These results are similar to those observed in our investigations of lignin peroxidase mediated oxidation of oxalate solubilize coal macromolecule. Previous studies in our laboratory using a spectrophotometric assay suggested that, in addition to oxalate, several other fungal metabolites are able to solubilize leonardite. We have reinvestigated this phenomenon using a more reliable gravimetric procedure for assessing solubilization. Our results confirm our earlier findings that malate, oxaloacetate and citrate are effective solubilizing agents whereas succinate, fumarate and α-ketoglutarate solubilize relatively small amounts of leonardite. Finally, we have studied the composition of the insoluble material remaining following extensive solubilization by sodium oxalate. The ratio of hydrogen to carbon is increased in the insoluble material relative to the parent leonardite. However, the ratio of oxygen to carbon is also increased in the insoluble material. Thus, the insoluble material does not appear to be more highly reduced that the parent leonardite and is not likely to be a better fuel that the parent material.
Portions of this document may be illegible in electronic image products. Images are produced from... more Portions of this document may be illegible in electronic image products. Images are produced from the best available original document. Disclaimer This rep rt was prepared as an a writ of work sponsored by an agency of the United States Government.
The wood-rotting fungus Phanerochaete chrysosporium is able to degrade a wide variety of environm... more The wood-rotting fungus Phanerochaete chrysosporium is able to degrade a wide variety of environmentally-persistent organic pollutants to carbon dioxide. The unique biodegradative abilities of this fungus are due, in part, to lignin peroxidases, oxidative enzymes that are secreted in response to nutrient deprivation. Lignin peroxidases catalyze the initial oxidation of many of the organic pollutants that are degraded by this fungus. They also mediate the initial oxidation of N,N,N{prime},N{prime},N{double_prime},N{double_prime}-hexamethylpararosaniline, several azo dyes and certain polycyclic aromatic hydrocarbons. Lignin peroxidases also mediate oxidative dechlorination. For example, lignin peroxidases oxidize pentachlorophenol to 2,3,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione. Similarly, these enzymes mediate oxidative oligomerization of 4-chloroaniline, resulting in production of several dimers, trimers and tetramers and net dechlorination of the aromatic ring. Interestingly, m...
Boiling Air Force Base, D.C. 20332-6448 AvDilability Cedp l j~~tst ipecinI 92-30010 I iEIIelligYl... more Boiling Air Force Base, D.C. 20332-6448 AvDilability Cedp l j~~tst ipecinI 92-30010 I iEIIelligYli[. 9'2_. 1 2-.,5 2
The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hy... more The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distillation product obtained from coal tar) was demonstrated. Analysis by capillary gas chromatography and high-performance liquid chromatography showed that at least 22 PAHs, including all of the most abundant PAH components present in anthracene oil, underwent 70 to 100% disappearance during 27 days of incubation with nutrient nitrogen-limited cultures of this fungus. Because phenanthrene is the most abundant PAH present in anthracene oil, this PAH was selected for further study. In experiments in which [14C]phenanthrene was incubated with cultures of P. chrysosporium containing anthracene oil for 27 days, it was shown that 7.7% of the recovered radiolabeled carbon originally present in [14C]phenanthrene was metabolized to 14CO2 and 25.2% was recovered from the aqueous fraction, while 56.1 and 11.0% were recovered from the methyl...
A straightforward procedure using density functional theory (M06 2X) coupled with a group-equival... more A straightforward procedure using density functional theory (M06 2X) coupled with a group-equivalent approach is described that was used to calculate gas-phase heat of formation (Δf H°g,298) values for buckminsterfullerene (C60), C70 fullerene (C70), corannulene, coronene, and sumanene. This procedure was also used to calculate exceptionally accurate Δf H°g,298 values for a variety of single-ring aromatic and 2-7 ring polycyclic aromatic hydrocarbons (PAHs) as well as a large selection of other hydrocarbons and phenols. The approach described herein is internally consistent, and results for C60, C70, corannulene, coronene, and sumanene are in very close agreement with results reported by others who used higher-level computational theory. Statistical analysis of a test set containing benzene and 18 two to seven ring PAHs demonstrated that by using this approach a mean absolute deviation (MAD) and a root-mean-square deviation (RMSD) of 0.8 and 1.3 kJ/mol, respectively, were achieved for reference/experimental Δf H°g,298 values versus calculated/predicted Δf H°g,298 values. For statistical analysis of a larger test set containing 235 aromatic and aliphatic hydrocarbons and phenols, a MAD and a RMSD of 1.2 and 1.9 kJ/mol, respectively, were achieved for reference/experimental Δf H°g,298 values versus calculated/predicted Δf H°g,298 values.
Microsomes, Drug Oxidations and Chemical Carcinogenesis, 1980
Cytochrome P-450 and NADPH cytochrome c (P-450) reductase were purified from bovine adrenocortica... more Cytochrome P-450 and NADPH cytochrome c (P-450) reductase were purified from bovine adrenocortical (BAC) microsomes. Cytochrome P-450 AM-1 purified to 10–12 nanomoles P-450/mg protein by AM 2 SO 4 fractionation and hydrophobic interaction chromatography showed essentially one band by SDS-page (>95%) with a MW = 52,000. Fraction P-450 AM-2 with a specific content of 3-4 nanomoles P-450/mg protein contained two hemeproteins with M.W. of 49,000 and 52,000. Partially purified reductase reduced cytochrome P-450. Adrenodoxin was not required nor did it stimulate reduction. Photoaffinity labeling studies show the inhibitor derived label 1-(4-azidophenyl)imidazole to have high affinity for BAC microsomal P-450. Labeling experiments using partially purified BAC microsomal P-450 afforded stoichiometric label incorporation.
Al (noting that the initial death toll was reported by state-owned television as 3,934, at the sa... more Al (noting that the initial death toll was reported by state-owned television as 3,934, at the same time that some government media was indicating it could be 10,000 and the Thai Foreign Minister indicated it could be 15,000 after meeting with Myanmar's ambassador).
Uploads
Papers by John Bumpus