Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 ± 14 min⁻¹, and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions.     

Oxidative degradation of phenanthrene by the ligninolytic fungus Phanerochaete chrysosporium.

The ligninolytic fungus Phanerochaete chrysosporium oxidized phenanthrene and phenanthrene-9,10-quinone (PQ) at their C-9 and C-10 positions to give a ring-fission product, 2,2'-diphenic acid (DPA), which was identified in chromatographic and isotope dilution experiments. DPA formation from phenanthrene was somewhat greater in low-nitrogen (ligninolytic) cultures than in high-nitrogen (nonligninolytic) cultures and did not occur in uninoculated cultures. The oxidation of PQ to DPA involved both fungal and abiotic mechanisms, was unaffected by the level of nitrogen added, and was significantly faster than the cleavage of phenanthrene to DPA. Phenanthrene-trans-9,10-dihydrodiol, which was previously shown to be the principal phenanthrene metabolite in nonligninolytic P. chrysosporium cultures, was not formed in the ligninolytic cultures employed here. These results suggest that phenanthrene degradation by ligninolytic P. chrysosporium proceeds in order from phenanthrene----PQ----DPA, involves both ligninolytic and nonligninolytic enzymes, and is not initiated by a classical microsomal cytochrome P-450. The extracellular lignin peroxidases of P. chrysosporium were not able to oxidize phenanthrene in vitro and therefore are also unlikely to catalyze the first step of phenanthrene degradation in vivo. Both phenanthrene and PQ were mineralized to similar extents by the fungus, which supports the intermediacy of PQ in phenanthrene degradation, but both compounds were mineralized significantly less than the structurally related lignin peroxidase substrate pyrene was.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Identification of metabolites from phenanthrene oxidation by phenoloxidases and dioxygenases of Polyporus sp. S133

Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Biodegradation of 2,4,6-trinitrotoluene byPhanerochaete chrysosporium: Identification of initial degradation products and the discovery of a TNT metabolite that inhibits lignin peroxidases

Biodegradation of 2,4,6-trinitrotoluene (TNT) by the wood-rotting BasidiomycetePhanerochaete chrysosporium was studied in a fixed-film silicone membrane bioreactor and in agitated pellected cultures. The initial intermediate products of TNT biodegradation were shown to be 2-amino-4,6-dinitrotoluene (2amDNT) and 4-amino-2,6-dinitrotoluene (4amDNT). These intermediates were also degraded byP. chrysosporium. However, their rates of degradation were slow and appeared to represent rate-limiting steps in TNT degradation. The fact that 2amDNT and 4amDNT were further degraded is of importance. In most other microbial systems these compounds are typically not further degraded or are dimerized to even more persistent azo and azoxydimers. Similar to previous studies performed in stationary cultures, it was shown that substantial amounts of [¹⁴C]-TNT were degrade to [¹⁴C]-carbon dioxide in agitated pelleted cultures. Lignin peroxidase activity (assayed by veratryl alcohol oxidation) virtually disappeared upon addition of TNT to ligninolytic cultures ofP. chrysosporium. However, TNT, 2amDNT, and 4amDNT did not inhibit lignin peroxidase activity, nor were they substrates for this enzyme. Subsequent studies revealed that 4-hydroxylamino-2,6-dinitrotoluene, an intermediate in TNT reduction, was a potent lignin peroxidase inhibitor. Further studies revealed that this compound was also a substrate for lignin peroxidase H8.     

Enzymatic characterization of Chilean native wood-rotting fungi for potential use in the bioremediation of polluted environments with chlorophenols

This work presents a preliminary report of a series of studies on the ability of several indigenous wood-rotting fungi from Chile to produce hydrolytic and ligninolytic enzymes and the evaluation of these native microorganism to future research on potential applications in bioremediation programs. Wood-rotting Basidiomycete fungi were collected from indigenous hardwood forest in the South of Chile. Twenty-eight strains were identified and qualitative enzymatic tests for peroxidases, laccase, tyrosinase, xylanase and cellulase production were performed in solid medium. Eleven selected strains were evaluated in liquid medium to quantify their ligninolytic enzyme production and their capacity to grow in solid medium supplemented with 2,4-dichlorophenol (2,4-DCF), 2,4,6-trichlorophenol (2,4,6-TCF) and pentachlorophenol (PCP). PCP degradation and ligninolytic enzymes production were also evaluated in liquid medium. Results showed that laccase was present in 28 of the selected strains (≈73%). Peroxidase was present in 40% and cellulase in 37% of the strains. Xilanase and tyrosinase were obtained in a smaller percentage in the strains (28% and 7%, respectively). The 11 selected strains showed high concentrations of lignin peroxidase (Lip) and manganese peroxidase (MnP). Anthracophyllum discolor (Sp4), produced LiP and MnP at 90.3 and MnP 125.5 U L⁻¹ respectively, compared to the control fungus Phanerochaete chrysosporium CECT-2798 that produced 58.1 and 118.4 U L⁻¹ of LiP and MnP. Tolerance test showed that native Chilean fungi did not present high tolerance to 2,4,6-TCF and PCP but were quite tolerant to 25 and 50 mg L⁻¹ of 2,4-DCF. However, pre-acclimatization in 2,4-DCP notably improved the growth in medium with 2,4,6-TCP and PCP. PCP in liquid medium was efficiently degraded by the fungi Anthracophyllum discolor, Lenzites betulina (Ru-30) and Galerina patagónica (Sp3), and the major MnP activity was produced by A. discolor (Sp4) (67 U L⁻¹).     

Effect of soya lecithin on the enzymatic system of the white-rot fungi Anthracophyllum discolor

The present work optimized the initial pH of the medium and the incubation temperature for ligninolytic enzymes produced by the white-rot fungus Anthracophyllum discolor. Additionally, the effect of soya lecithin on mycelial growth and the production of ligninolytic enzymes in static batch cultures were evaluated. The critical micelle concentration of soya lecithin was also studied by conductivity. The effects of the initial pH (3, 4, and 5) and incubation temperature (20, 25, and 30°C) on different enzymatic activities revealed that the optimum conditions to maximize ligninolytic activity were 26°C and pH 5.5 for laccase and manganese peroxidase (MnP) and 30°C and pH 5.5 for manganese-independent peroxidase (MiP). Under these culture conditions, the maximum enzyme production was 10.16, 484.46, and 112.50 U L⁻¹ for laccase, MnP, and manganese-independent peroxidase MiP, respectively. During the study of the effect of soya lecithin on A. discolor, we found that the increase in soya lecithin concentration from 0 to 10 g L⁻¹ caused an increase in mycelial growth. On the other hand, in the presence of soya lecithin, A. discolor produced mainly MnP, which reached a maximum concentration of 30.64 ± 4.61 U L⁻¹ after 25 days of incubation with 1 g L⁻¹ of the surfactant. The other enzymes were produced but to a lesser extent. The enzymatic activity of A. discolor was decreased when Tween 80 was used as a surfactant. The critical micelle concentration of soya lecithin calculated in our study was 0.61 g L⁻¹.     

Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): studies on the effects of monooxygenase inducers and role of cytochromes P-450

The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (>90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography—mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with β-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55–60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.     

In vivo decolourization of the polymeric dye poly R‐478 by corncob cultures of Phanerochaete chrysosporium

In this paper, the in vivo decolourization of the polymeric dye Poly R‐478 by semi‐solid‐state cultures of Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) was investigated, employing corncob as a support. In order to stimulate the ligninolytic system of the fungus, the cultures were supplemented with veratryl alcohol (2 mM) or manganese (IV) oxide (1 g/l). Maximum manganese‐dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of around 2,000 U/l and 400 U/l were attained by the former, whereas the activities reached by the latter were of about 1,500 U/l and 200 U/l, respectively. Furthermore, laccase activity (around 150 U/l) was only detected in manganese (IV) oxide supplemented cultures. The polymeric dye Poly R‐478 (0.02 w/v) was added to three‐day‐old cultures. A percentage of biological decolourization of about 85% was achieved using cultures supplemented with veratryl alcohol, whereas MnO₂ cultures showed a rather lower percentage of around 58% after nine days of dye incubation. Moreover, a correlation between MnP activity and Poly R‐478 decolourization could be observed, indicating that this enzyme is mainly responsible for dye degradation. In the present work, the in vivo decolourizing capability of the ligninolytic complex secreted by P. chrysosporium was investigated under the above‐mentioned cultivation conditions, employing a model compound, such as the polymeric dye Poly R‐478.     

Degradation of 4-nitrophenol by the lignin-degrading basidiomycete <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The fungal metabolism of 4-nitrophenol (4-NP) was investigated using the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Despite its phenolic feature, 4-NP was not oxidized by extracellular ligninolytic peroxidases. However, 4-NP was converted to 1,2-dimethoxy-4-nitrobenzene via intermediate formation of 4-nitroanisole by the fungus only under ligninolytic conditions. The metabolism proceeded via hydroxylation of the aromatic ring and methylation of phenolic hydroxyl groups. Although the involvement of nitroreductase in the metabolism of 2,4-dinitrotoluene by many aerobic and anaerobic microorganisms including P. chrysosporium has been reported, no formation of 4-aminophenol was observed during 4-NP metabolism. The formation of 1,2-dimethoxy-4-nitrobenzene was effectively inhibited by exogenously added piperonyl butoxide, a cytochrome P450 inhibitor, suggesting that cytochrome P450 is involved in the hydroxylation reaction. Thus, P. chrysosporium seems to utilize hydroxylation and methylation reactions to produce a more susceptible structure for an oxidative metabolic system.     

Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P-450

Degradation of 2,4,6-trinitrotoluene (TNT) by the white-rot fungus Bjerkandera adusta DSM 3375 was studied in relation to extracellular ligninolytic activities. The Mn(II)-dependent peroxidase, the only ligninolytic enzyme detectable, reached a maximum activity of 600 ± 159 U/l after incubation in mineral medium with a sufficient nitrogen source. In contrast, the highest extent of [¹⁴C]TNT mineralization was detected in malt extract broth, so that the ability of B. adusta to mineralize TNT did not parallel ligninolytic activity. The microsomal fraction of cells grown in the presence of TNT was found to contain 11 pmol cytochrome P-450/mg protein. In cells grown without TNT, no microsomal cytochrome P-450 could be found. Instead, 14 pmol P-450/mg protein was present in the cytosolic fraction of these cells. Cytochrome P-450 apparently affected the TNT metabolism, as shown by inhibitory studies. Addition of the cytochrome P-450 inhibitor piperonyl butoxide diminished the ¹⁴CO₂ release from 21% to 0.9%, as determined after 23 days of incubation, while 1-aminobenzotriazole and metyrapone decreased the mineralization to 8.6% and 6.3% respectively. Mass-balance analysis of TNT degradation in liquid cultures revealed that, by inhibition of cytochrome P-450, the TNT-derived radioactivity associated with biomass and with polar, water-soluble metabolites decreased from 93.9% to 15.0% and the fraction of radiolabelled metabolites extractable with organic solvents fell to 92.6%. The TNT metabolites of this fraction were identified as aminodinitrotoluenes, indicating that this initial transformation product of TNT may function as a substrate for cytochrome-P-450-dependent reactions in B. adusta. Received: 27 May 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999     

Degrading ability of oligocyclic aromates by <Emphasis Type="Italic">Phanerochaete sordida</Emphasis> selected <Emphasis Type="Italic">via</Emphasis> screening of white rot fungi

Seventy-nine white rot strains were screened to determine if they had the potential for use in the degradation of oligocyclic aromates (PAHs) by measuring their dye-decoloration rate. Fourteen strains that were selected based on their dye-decoloration rate were then evaluated for the ability to tolerate various levels of PAHs spiked in agar medium. The ability of white rot fungi to degrade 3- or 4-ring PAHs (anthracene, phenanthrene, fluoranthene, pyrene) was determined. Two strains of Phanerochaete sordida (KUC8369, KUC8370) were possible PAHs degraders, degrading a significantly greater amount of phenanthrene and fluoranthene than the culture collection strain P. chrysosporium (a known PAHs degrader). The production of manganese peroxidase, the only extracellular ligninolytic enzyme detected during the cultivation, was evaluated.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens

The metabolism of phenanthrene, a polycyclic aromatic hydrocarbon (PAH), by Streptomyces flavovirens was investigated. When grown for 72 h in tryptone yeast extract broth saturated with phenanthrene, the actinomycete oxidized 21.3% of the hydrocarbon at the K-region to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol). A trace of 9-phenanthrol was also detected. Metabolites isolated by thin-layer and high performance liquid chromatography were identified by comparing chromatographic, mass spectral, and nuclear magnetic resonance properties with those of authentic compounds. Experiments using [9-¹⁴C]phenanthrene showed that the trans-9,10-dihydrodiol had 62.8% of the radioactivity found in the metabolites. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was (–)-9S,10S, the same as that of the principal enantiomer produced by mammalian enzymes. Incubation of S. flavovirens with phenanthrene is an atmosphere of ¹⁸O₂, followed by gas chromatographic/mass spectral analysis of the metabolites, indicated that one atom from molecular oxygen was incorporated into each molecule of the phenanthrene trans-9,10-dihydrodiol. Cytochrome P-450 was detected in 105,000×g supernatants prepared from cell extracts of S. flavovirens. The results show that the oxidation of phenanthrene by S. flavovirens was both regio- and stereospecific.Abbreviations CD circular dichroism - DMF N,N-dimethyl-formamide - GC/MS gas chromatography/mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - ODS octadecylsilane - PAH polycyclic aromatic hydrocarbon - TLC thin-layer chromatography - TMS tetramethylsilane - UV ultraviolet     

Metabolism of phenanthrene by the white rot fungus Pleurotus ostreatus.

The white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene, metabolized 94% of the phenanthrene added. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized to trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) (28%), 2,2'-diphenic acid (17%), and unidentified metabolites (7%). Nonextractable metabolites accounted for 35% of the total radioactivity. The metabolites were extracted with ethyl acetate, separated by reversed-phase high-performance liquid chromatography, and characterized by 1H nuclear magnetic resonance, mass spectrometry, and UV spectroscopy analyses. 18O2-labeling experiments indicated that one atom of oxygen was incorporated into the phenanthrene trans-9,10-dihydrodiol. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was 9R,10R, which is different from that of the principal enantiomer produced by Phanerochaete chrysosporium. Significantly less phenanthrene trans-9,10-dihydrodiol was observed in incubations with the cytochrome P-450 inhibitor SKF 525-A (77% decrease), 1-aminobenzotriazole (83% decrease), or fluoxetine (63% decrease). These experiments with cytochrome P-450 inhibitors and 18O2 labeling and the formation of phenanthrene trans-9R,10R-dihydrodiol as the predominant metabolite suggest that P. ostreatus initially oxidizes phenanthrene stereoselectively by a cytochrome P-450 monoxygenase and that this is followed by epoxide hydrolase-catalyzed hydration reactions.     

Comparison of two lignin-degrading fungi:Phlebia radiata andPhanerochaete chrysosporium

Summary The ligninolytic enzymes ofPhlebia radiata were produced in static conditions earlier developed forPhanerochaete chrysosporium. The production pattern of lignin peroxidases resembled that ofP. chrysosporium. The extracellular proteins ofPhlebia radiata were separated by isoelectric focusing. Four proteins with acidic isoelectric points (4.15) were detected by peroxidase staining. The peroxidases ofP. radiata reacted with antibodies produced against a peroxidase ofPhanerochaete chrysosporium and vice versa. Thus the lignin peroxidases of the two fungi have major similarities despite slight differences in their isoelectric points and molecular weights. Veratryl alcohol was produced by both fungi and degraded to veratraldehyde, two lactones and a quinone by the ligninolytic cultures.     

Production of the ligninolytic enzymes by immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in an air atmosphere

Summary The production of the ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in air was investigated by adopting different sizes and amounts of the carriers, different medium C/N ratios and different glucose-feeding strategies. No lignin peroxidase (LiP) activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NH₄⁺, 56/2.2 mM) with two sizes and three amounts of the carriers, while comparable levels of manganese peroxidase (MnP) activities were detected only in non-immersed cultures with two sizes of the carriers. A non-immersed state also stimulated LiP formation under carbon limitation (C/N ratio 28/44 mM). High peak activities of LiP, 197 and 164 U/l, were obtained in non-immersed cultures under carbon limitation at the C/N ratios of 28/44 and 56/44 mM, respectively, the occurrence of the activities coinciding with the complete consumption of glucose. A very low level of MnP was measured at the C/N ratio of 28/44 mM compared with the similar activities at 56/2.2 and 56/44 mM. An addition of 2 g glucose/l after its complete depletion improved both the production of LiP and MnP markedly in non-immersed culture at the initial C/N ratio of 28/44 mM, whereas a replenishment of 5 g/l, still enhancing the formation of MnP, inhibited the production of LiP first before the later reactivation. It is suggested that non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy is one potential way to realize high production of the ligninolytic enzymes by P. chrysosporium in air.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.