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PubMed1993

Degradation of benzene, toluene, ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Degradation of the BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylenes) group of organopollutants by the white-rot fungus Phanerochaete chrysosporium was studied. Our results show that the organism efficiently degrades all the BTEX components when these compounds are added either individually or as a composite mixture. Degradation was favored under nonligninolytic culture conditions in malt extract medium, in which extracellular lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) are not produced. The noninvolvement of LIPs and MNPs in BTEX degradation was also evident from in vitro studies using concentrated extracellular fluid containing LIPs and MNPs and from a comparison of the extents of BTEX degradation by the wild type and the per mutant, which lacks LIPs and MNPs. A substantially greater extent of degradation of all the BTEX compounds was observed in static than in shaken liquid cultures. Furthermore, the level of degradation was relatively higher at 25 than at 37 degrees C, but pH variations between 4.5 and 7.0 had little effect on the extent of degradation. Studies with uniformly ring-labeled [14C]benzene and [14C]toluene showed substantial mineralization of these compounds to 14CO2.

Animals↗
PubMed1993

Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium rapidly mineralizes 2,4,5-trichlorophenol. The pathway for degradation of 2,4,5-trichlorophenol was elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway involves cycles of peroxidase-catalyzed oxidative dechlorination reactions followed by quinone reduction reactions to yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably ring cleaved. In the first step of the pathway, 2,4,5-trichlorophenol is oxidized to 2,5-dichloro-1,4-benzoquinone by either MnP or Lip. 2,5-Dichloro-1,4-benzoquinone is then reduced to 2,5-dichloro-1,4-hydroquinone. The 2,5-dichloro-1,4-hydroquinone is oxidized by MnP to generate 5-chloro-4-hydroxy-1,2-benzoquinone. The orthoquinone is in turn reduced to 5-chloro-1,2,4-trihydroxybenzene. Finally, the 5-chlorotrihydroxybenzene undergoes another cycle of oxidative dechlorination and reduction reactions to generate 1,2,4,5-tetrahydroxybenzene. The latter is presumably ring cleaved, with subsequent degradation to CO2. In this pathway, the substrate is oxidatively dechlorinated by LiP or MnP in a reaction which produces a quinone. The quinone intermediate is recycled by a reduction reaction to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This pathway apparently results in the removal of all three chlorine atoms before ring cleavage occurs.

Basidiomycota↗
PubMed1993

Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium.

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.

Amino Acid Sequence↗
PubMed1995

Transformation of (sup14)C-Lignin-Labeled Cell Walls of Wheat by Syntrophococcus sucromutans, Eubacterium oxidoreducens, and Neocallimastix frontalis.

Wheat cell walls, saponified or not, labeled with [U-(sup14)C]phenylalanine or [O-methyl-(sup14)C]sinapate were fermented by Neocallimastix frontalis or Syntrophococcus sucromutans plus Eubacterium oxidoreducens or a mixed culture. Phenolics were less solubilized but more transformed by bacteria than by the fungus, and mineralization was slight. S. sucromutans O-demethylated [O-methyl-(sup14)C]syringyl lignins, yielding labeled acetate.

Journal Article↗
PubMed1995

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters.

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.

Journal Article↗
PubMed1997

Lignin Peroxidase Isozymes from Phanerochaete chrysosporium Can Be Enzymatically Dephosphorylated.

The extracellular lignin peroxidase (LIP) protein profile of the fungus Phanerochaete chrysosporium, grown in nonimmersed liquid culture under conditions of excess nitrogen, changed markedly with culture age. At peak LIP activity (day 4), the heme-protein profile in the extracellular fluid, analyzed by anion-exchange high-pressure liquid chromatography, was characterized by a predominance of the LIP isozymes H1 and H2, small amounts of H6 and H8, and other minor peaks, designated Ha and Hb. On day 5, the level of H1 increased and it became the dominant isozyme, with a corresponding decrease in the level of H2. Moreover, the relative levels of H6 and H8 decreased with corresponding increases in Ha and Hb levels. This change in LIP profile occurred extracellularly and resulted from the enzymatic dephosphorylation of LIP isozymes. An enzymatic fraction responsible for LIP isozyme dephosphorylation, termed LIP dephosphorylating (LpD) fraction, was partially purified from the culture fluid. Incubation of the LpD fraction with (sup32)P-labeled H2, H6, H8, and H10 isozymes separated from nitrogen-limited cultures resulted in the formation of the dephosphorylated isozymes H1, Ha, Hb, and Hc, respectively. Dephosphorylation did not significantly change the catalytic properties of the LIP isozymes with veratryl alcohol as a substrate. LIP dephosphorylation is therefore suggested to be a posttranslational modification process catalyzed extracellularly by the LpD activity.

Journal Article↗
PubMed1997

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.

Basidiomycota↗
PubMed1998

2-chloro-1,4-dimethoxybenzene as a novel catalytic cofactor for oxidation of anisyl alcohol by lignin peroxidase.

2-Chloro-1,4-dimethoxybenzene (2Cl-14DMB) is a natural compound produced de novo by several white rot fungi. This chloroaromatic metabolite was identified as a cofactor superior to veratryl alcohol (VA) in the oxidation of anisyl alcohol (AA) by lignin peroxidase (LiP). Our results reveal that good LiP substrates, such as VA and tryptophan, are comparatively poor cofactors in the oxidation of AA. Furthermore, we show that a good cofactor does not necessarily serve a role in protecting LiP against H(2)O(2) inactivation. 2Cl-14DMB was not a direct mediator of AA oxidation, since increasing AA concentrations did not inhibit the oxidation of 2Cl-14DMB at all. However, the high molar ratio of anisaldehyde formed to 2Cl-14DMB consumed, up to 13:1, indicates that a mechanism which recycles the cofactor is present.

Journal Article↗
PubMed1998

Expression of phanerochaete chrysosporium genes encoding lignin peroxidases, manganese peroxidases, and glyoxal oxidase in wood

Expression of Phanerochaete chrysosporium genes encoding ligninolytic enzymes was assessed in wood. Poly(A) RNA was extracted from colonized wood chips by magnetic capture, and specific transcripts were quantified by competitive reverse transcriptase PCR. mRNA levels varied substantially among lignin peroxidase genes, and transcript patterns were dramatically different from those in previous studies with defined media.

Journal Article↗
PubMed1999

Manganese deficiency can replace high oxygen levels needed for lignin peroxidase formation by Phanerochaete chrysosporium.

The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.

Benzyl Alcohols↗
PubMed1999

Homologous expression of recombinant lignin peroxidase in Phanerochaete chrysosporium.

The glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter was used to drive expression of lip2, the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium. The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura+ transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.

Genetic Vectors↗
PubMed2003

Reactive oxygen species and induction of lignin peroxidase in Phanerochaete chrysosporium.

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.

Culture Media↗
PubMed1992

Lignin peroxidase gene family of Phanerochaete chrysosporium: complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome.

Lignin peroxidases (LiP) of Phanerochaete chrysosporium are encoded by a family of six closely related genes. Five LiP genes have been localized to the same dimorphic chromosome. In this investigation, relative transcript levels of the LiP genes were determined. Transcripts of the LiPA, LiPB, and O282 genes were at similar levels in both carbon- and nitrogen-limited cultures. In contrast, transcription of the GLG5, V4, and GLG4 genes was dramatically altered by culture conditions. Under carbon-limited conditions, GLG4 transcripts were, by far, the most abundant. Southern blot analyses of clamped homogeneous field gels were used to map the GLG4 gene to a dimorphic chromosome separate from the other LiP genes.

Base Sequence↗
PubMed1992

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.

Basidiomycota↗
PubMed1993

Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium.

Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA.

Cell Compartmentation↗
PubMed1999

Organization and differential regulation of a cluster of lignin peroxidase genes of Phanerochaete chrysosporium.

The lignin peroxidases of Phanerochaete chrysosporium are encoded by a minimum of 10 closely related genes. Physical and genetic mapping of a cluster of eight lip genes revealed six genes occurring in pairs and transcriptionally convergent, suggesting that portions of the lip family arose by gene duplication events. The completed sequence of lipG and lipJ, together with previously published sequences, allowed phylogenetic and intron/exon classifications, indicating two main branches within the lip family. Competitive reverse transcription-PCR was used to assess lip transcript levels in both carbon- and nitrogen-limited media. Transcript patterns showed differential regulation of lip genes in response to medium composition. No apparent correlation was observed between genomic organization and transcript levels. Both constitutive and upregulated transcripts, structurally unrelated to peroxidases, were identified within the lip cluster.

Carbon↗
PubMed1989

Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium.

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).

Amino Acid Sequence↗