White Rot Fungus Phanerochaete Chrysosporium Research Articles

Ligninolytic and Nonligninolytic Mineralization of Trinitrotoluene by Several White Rot Basidiomycetes

ABSTRACT The explosive 2,4,6-trinitrotoluene (TNT) is considered a toxic environmental pollutant that contaminates the soil and ground water. The white rot fungus Phanerochaete chrysosporium is well known for the degradation of TNT under ligninolytic condition. Very few, if any, studies have been done using other white rot fungi. In this study four fungal species, namely, P. chrysosporium, Kuehneromyces mutabilis, Hypholoma fasciculare, and Phlebia radiata, were used to investigate TNT degradation. All fungi were grown under ligninolytic (low-nitrogen) and nonligninolytic (high-nitrogen) conditions containing 25 parts per million (ppm) (0.11 mM) of TNT. Analysis by high-performance liquid chromatography (HPLC) showed biotransformation of TNT under both conditions. Complete degradation occurred under ligninolytic conditions (peroxidase enzymes were present) by P. chrysosporium and P. radiata. A nitrite release assay at 6 days indicated the denitrifying abilities of all the tested varieties of white rot fungi. For both ligninolytic and non-ligninolytic conditions, mass-balance studies showed biotransformation of 0.5 μ Ci 14C-labeled TNT with pregrown mycelial pellets of all fungal species, in which 5% to 15% of the TNT was converted to CO2. These studies show that TNT may be degraded by several other species of white rot fungi and provided additional information on the biodegradation of nitroaromatic compounds in the environment.

Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors

The cellobiohydrolase Pc_Cel7D is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium, constituting approximately 10% of the total secreted protein in liquid culture on cellulose. The enzyme is classified into family 7 of the glycoside hydrolases and, like other family members, catalyses cellulose hydrolysis with net retention of the anomeric carbon configuration. Previous work described the apo structure of the enzyme. Here we investigate the binding of the product, cellobiose, and several inhibitors, i.e. lactose, cellobioimidazole, Tris/HCl, calcium and a thio-linked substrate analogue, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (GG-S-GG). The three disaccharides bind in the glucosyl-binding subsites +1 and +2, close to the exit of the cellulose-binding tunnel/cleft. Pc_Cel7D binds to lactose more strongly than cellobiose, while the opposite is true for the homologous Trichoderma reesei cellobiohydrolase Tr_Cel7A. Although both sugars bind Pc_Cel7D in a similar fashion, the different preferences can be explained by varying interactions with nearby loops. Cellobioimidazole is bound at a slightly different position, displaced approximately 2 A toward the catalytic centre. Thus the Pc_Cel7D complexes provide evidence for two binding modes of the reducing-end cellobiosyl moiety; this conclusion is confirmed by comparison with other available structures. The combined results suggest that hydrolysis of the glycosyl-enzyme intermediate may not require the prior release of the cellobiose product from the enzyme. Further, the structure obtained in the presence of both GG-S-GG and cellobiose revealed electron density for Tris at the catalytic centre. Inhibition experiments confirm that both Tris and calcium are effective inhibitors at the conditions used for crystallization.

Complete Detoxification of Olive Mill Wastewaters by Integrated Treatment Using the White Rot Fungus Phanerochaete chrysosporium Followed by Anaerobic Digestion and Ultrafiltration

Complete Detoxification of Olive Mill Wastewaters by Integrated Treatment Using the White Rot Fungus Phanerochaete chrysosporium Followed by Anaerobic Digestion and Ultrafiltration

Phenol Metabolism by White Rot Fungus Phanerochaete chrysosporium Isolated from Indian Paper Mill Effluent Enriched Soil Samples

Phenol Metabolism by White Rot Fungus Phanerochaete chrysosporium Isolated from Indian Paper Mill Effluent Enriched Soil Samples

Decolorization and Degradation of Phenolic Paper Mill Effluent by Native White Rot Fungus Phanerochaete chrysosporium

Decolorization and Degradation of Phenolic Paper Mill Effluent by Native White Rot Fungus Phanerochaete chrysosporium

Fungal degradation of wood: initial proteomic analysis of extracellular proteins of Phanerochaete chrysosporium grown on oak substrate

Two-dimensional (2-D) gel electrophoresis was used to separate the extracellular proteins produced by the white-rot fungus Phanerochaete chrysosporium. Solid-substrate cultures grown on red oak wood chips yielded extracellular protein preparations which were not suitable for 2-D gel analysis. However, pre-washing the wood chips with water helped decrease the amount of brown material which caused smearing on the acidic side of the isoelectric focusing gel. The 2-D gels from these wood-grown cultures revealed more than 45 protein spots. These spots were subjected to in-gel digestion with trypsin followed by either peptide fingerprint analysis by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or by liquid chromatography (LC)/MS/MS sequencing. Data from both methods were analyzed by Protein Prospector and the local P. chrysosporium annotated database. MALDI-TOF/MS only identified two proteins out of 25 analyzed. This was most likely due to problems associated with glycosylation. Protein sequencing by LC/MS/MS of the same 25 proteins resulted in identification of 16 proteins. Most of the proteins identified act on either cellulose or hemicellulose or their hydrolysis products. Thus far no lignin peroxidase, Mn peroxidase or laccases have been detected.

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1→3)-β-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1→3)-β-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6- O-glucosyl-laminaribiose (β- d-Glc p-(1→6)-β- d-Glc p-(1→3)- d-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis–Menten equation including hydrolytic and transglycosylation parameters ( K m, K m2, k cat, k cat2). The rate of 6- O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K m, k cat, and affinity for glucosyl-enzyme intermediates ( K m2) were estimated for laminarioligosaccharides (DP = 3–5), the K m value of laminaribiose was approximately 5–9 times higher than those of the other oligosaccharides (DP = 3–5), whereas the K m2 values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters ( K m and k cat), and a possible mechanism of transglycosylation is proposed.

Differential regulation and xenobiotic induction of tandem P450 monooxygenase genes pc-1 (CYP63A1) and pc-2 (CYP63A2) in the white-rot fungus Phanerochaete chrysosporium.

The two tandem P450 monooxygenase genes (pc-1 and pc-2) reported by us earlier in Phanerochaete chrysosporium were investigated for their regulation under nutrient-limited and nutrient-rich culture conditions. Transcript analysis based on real-time quantitative RT-PCR showed higher expression of pc-1 in defined low-nitrogen and pc-2 in defined high-nitrogen media, with maximum expression on day 4, indicating that the two genes, though tandemly linked, are regulated in a non-coordinate manner. Transcript levels of pc-1 and pc-2 were differentially influenced by the type of carbon source, incubation temperature, and oxygenation. Both genes were inducible by organic xenobiotic chemicals. Of the 42 xenobiotics tested in nutrient-rich cultures, pc-1 transcription was induced 2.12(+/-0.40)-fold to 6.27(+/-0.48)-fold in the presence of 19 compounds and pc-2 transcription was induced 2.00(+/-0.73)-fold to 29.39(+/-9.40)-fold in the presence of 22 compounds. Particularly, it is significant that both pc-1 and pc-2 are induced by polycyclic aromatic hydrocarbons (PAHs) of varying ring size, including naphthalene (4.35+/-0.09, 6.02+/-1.39), phenanthrene (2.82+/-0.12, 2.14+/-0.61), pyrene (3.93+/-0.01, 1.0+/-0.12), benzanthracene (1.67+/-0.03, 6.08+/-1.50), and benzo(a)pyrene (1.55+/-0.01, 5.54+/-2.75) respectively. This study constitutes the first report on the identification of P450 genes in a fungus that are responsive to environmentally significant pollutant chemicals (PAHs, DDT, long-chain alkyl phenols) and lignin derivatives.

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN 3, ascorbic acid, β-mercaptoethanol and dithreitol. The K m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.

Molecular approach for analysis of model fungal genes encoding ligninolytic peroxidases in wood-decaying soil systems

Test the use of nondegenerated consensus polymerase chain reaction (PCR) primers targeting lip and mnp sequences to detect ligninolytic fungi in wood-decaying soil systems, avoiding the need for enrichment or isolation on traditional fungal media culture. The PCR primers were tested with total DNA isolated from incubations of wood-soil systems inoculated or not with the white-rot fungi Phanerochaete chrysosporium, or a white-rot sample obtained from a Nothofagus forest. The PCR products for lip and mnp sequences were only obtained in soil with P. chrysosporium-colonized wood chips. In these soil samples, reverse transcription-PCR analysis of lip and mnp PCR products indicated expression of LipA, LipB, LipJ and MnP isoenzymes. This is the first assessment of the use of consensus PCR primers for direct detection of ligninolytic peroxidase genes in wood-decaying soil systems.

Decolorization of sulfonphthalein dyes by manganese peroxidase activity of the white-rot fungus Phanerochaete chrysosporium.

Manganese peroxidase (MnP) was produced by shallow stationary cultures of Phanerochaete chrysosporium growing on N-limited medium. Decolorization of sulfonphthalein (SP) dyes by MnP was investigated. The MnP activity profile and decolorization of SP dyes was correlated and almost all dyes were decolorized at pH 4.0. The influence of various inhibitors on Bromocresol Purple decolorization suggested an oxidative nature of the MnP-catalyzed decolorization of SP dyes.

Vertebrate helentrons and other novel Helitrons

Helitrons, a novel class of eukaryote mobile genetic elements, are distinguished from other transposable elements by encoding a ‘rolling circle’ replication (RCR) protein (Rep) and a helicase. Helitrons have recently been described from Arabidopsis, rice and the nematode Caenorhabditis. We now report the discovery of Helitron-like elements in vertebrates, specifically in the genomes of the fish Danio rerio and Sphoeroides nephelus. We also describe Helitrons from the white rot fungus Phanerochaete chrysosporium and from the Anopheles genome. Many of the fish Helitrons have an uncorrupted open reading frame encoding both the RCR Rep protein and a helicase. These fish elements are of particular interest because they also encode, within the single open reading frame, an apurinic-apyrimidinic (AP) endonuclease most closely related to those of certain non-long terminal repeat retrotransposons. As they invariably carry an endonuclease and also form a very distinct clade, we have named these vertebrate elements ‘helentrons’. It is likely that these helentrons are still active.

Iron-responsive genes of Phanerochaete chrysosporium isolated by differential display reverse transcription polymerase chain reaction.

White-rot fungus Phanerochaete chrysosporium, a ligninolytic basidiomycete, was studied to identify iron-responsive genes. Using the differential display reverse transcription PCR technique (DDRT-PCR), a total of 97 differentially expressed cDNA fragments were identified by comparing band intensities among fingerprints obtained from mycelia cultivated in iron-deficient and iron-replete media. Transcripts induced under iron-starvation exhibited homologies to: a modular polyketide synthase, a TonB protein, a probable transmembrane protein, a putative ABC transporter permease and a HSP70-related heat-shock protein. Modular polyketide synthase and TonB proteins are normally expressed under iron-starvation and are known to be involved in biosynthesis and transport of siderophores respectively. Also, a deduced protein with 96% similarity to a precursor of the well-known P. chrysosporium lignin peroxidase was identified under iron-deficiency. Two DDRT-PCR products confirmed their iron-induced expression. One was homologue to the CNOT3, which is a global regulator of RNA polymerase II transcription and has been implicated in multiple roles in the control of mRNA metabolism. The other was similar to the Schizosaccharomyces pombe putative proteasome maturation factor upm1. In conclusion, the majority of iron-responsive P. chrysosporium transcripts isolated in the DDRT-PCR encode proteins involved in iron acquisition, especially members of biosynthesis and transport of iron chelators.

Treatment of Landfill Leachate by White Rot Fungus in Combination with Zeolite Filters

This article presents the experimental work for the treatment of landfill leachate in a combined process using the white rot fungus Phanerochaete chrysosporium and the natural zeolite Clinoptilolite. Clinoptilolite was used in a pretreatment step as a sink for ammonia nitrogen and, on average it reduced the levels of ammonia nitrogen, soluble chemical oxygen demand (COD) and color by 72, 4.7, and 25%, respectively. The reductions by fungal treatment alone were 16.6, 21.5, and 31.2%, respectively. However, a reduction in nitrogen loading greatly enhanced fungal treatment efficiency. A high C/N ratio in the leachate was found preferable for the fungal treatment. With the synergy created by pretreatment and fungal growth that was stimulated by the addition of a growth medium, the process could remove ammonia nitrogen, soluble COD (SCOD) and color at levels as high as 81.5, 65, and 59%, respectively. The ratio of SBOD5/SCOD increased from 0.1 to 0.17 upon treatment, indicating that the process rendered the leachate more amenable to the biological process. This result suggested that the preliminary reduction of ammonia nitrogen was essential in making the fungal process practicable for landfill leachate treatment.

Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris.

The extracellular beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-beta-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-beta-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.

Family 3 β-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-β-glucosidase

The substrate specificity of an extracellular β-glucosidase (BGL) from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium was investigated, using a variety of compounds with β-glucosidic linkages. Amino acid sequencing data for the purified BGL showed that the enzyme is identical to the glycoside hydrolase (GH) family 3 BGL of the same fungus previously reported [Li, B. and Renganathan, V, Appl. Environ. Microbiol., 64, 2748-2754 (1998)]. The BGL can hydrolyze both cellobiose and cellobionolactone, but cellobionolactone was hydrolyzed very much more slowly than cellobiose. Moreover, cellobionolactone inhibited cellobiose hydrolysis by the BGL, suggesting that this enzyme cannot cooperate with cellobiose dehydrogenase (CDH) in cellulose degradation by P. chrysosporium. In addition to cellobiose, BGL utilized various glucosyl-β-glucosides, such as sophorose, laminaribiose and gentiobiose, as substrates. Among the four substrates, laminaribiose (β-1,3-glucosidic linkage) was hydrolyzed most effectively. Moreover, the hydrolytic rate of laminarioligosaccharides increased proportionally to the degree of polymerization (DP), and the activity of BGL even towards laminarin with an average DP of 25 was similar to that towards laminaripentaose (DP 5). Therefore, we conclude that the extracellular BGL from P. chrysosporium is primarily a glucan 1,3-β-glucosidase (EC 3.2.1.58), which might play a role on fungal cell wall metabolism, rather than a β-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of β-1,4-glucosidic compounds during cellulose degradation.

Tandem cytochrome P450 monooxygenase genes and splice variants in the white rot fungus Phanerochaete chrysosporium: cloning, sequence analysis, and regulation of differential expression

Phanerochaete chrysosporium, the model white rot fungus, uses P450 monooxygenation for oxidation of xenobiotic chemicals. To characterize its P450 systems, we have isolated two tandemly linked full-length P450 monooxygenase genes, pc-1 (2510 bp) and pc-2 (2503 bp). cDNA sequence and splice site analysis showed multiple small putative introns (35–82 bp), 17 in pc-1 and 16 in pc-2. The deduced P450 proteins PC-1 (531 aa, MW 60,070) and PC-2 (538 aa, MW 61,765) showed 55% aa identity with each other and low relatedness (25–36%) with P450 gene families CYP52, CYP86, CYP4, and CYP3 decreasing in that order. Alternate splicing was identified for pc-1 transcription. Real-time quantitative RT-PCR analysis on 4-day cultures showed an elevated level of expression (∼8-fold) of pc-1 in nutrient-limited (ligninolytic) as compared to nutrient-rich (non-ligninolytic) conditions. Interestingly, the pc-1 gene is expressible in both primary and secondary phases of growth with peak expression occurring with secondary metabolism.

Manganese-containing superoxide dismutase from the white-rot fungus Phanerochaete chrysosporium: its function, expression and gene structure

The gene structure and expression of the antioxidant enzyme manganese-containing superoxide dismutase ( MnSOD1) of the white-rot fungus Phanerochaete chrysosporium was characterized. The MnSOD1 protein was purified from liquid cultures of P. chrysosporium and the amino acid sequences of four of its tryptic fragments were determined and used to clone a 3.2-kb genomic fragment encompassing the entire gene of MnSOD1. Sequence analysis of the new gene, MnSOD1, revealed more than 80% identity with the MnSOD genes from the white-rot fungi Ganoderma resinaceum, Ganoderma microsporum and Amauroderma rude, forming a clearly discernible cluster. The expression of MnSOD1 was measured as a function of culture age, and a good correlation between transcript level and enzyme activity was observed, with a peak at 72 and 96 h of incubation, respectively. Interestingly, as for P. chrysosporium, the only SOD activity detectable in linear cultures of other white-rot fungi tested ( Pleurotus florida, Lentinus edodes, Trametes versicolor, Ganoderma lucidum) was cyanide-insensitive SOD activity defined as MnSOD.

Starch-based plastic polymer degradation by the white rot fungus Phanerochaete chrysosporium grown on sugarcane bagasse pith: enzyme production.

In this study, starch metabolites and enzymes were determined during starch-based plastic polymer biodegradation by the white rot fungus Phanerochaete chrysosporium, grown in sugarcane bagasse pith in tubular reactors. Various metabolites, amylase, ligninase and cellulase production were measured during P. chrysosporium growth on sugarcane bagasse pith with added glucose and starch polymer. On-line respirometric analyses followed during 32 days confirmed the P. chrysosporium capability of growing on sugarcane bagasse pith with starch polymer degradation. Enzyme activity during secondary metabolism increased, and a 70% and 74% starch degradation was reached with and without glucose addition, generating low molecular weight metabolites (e.g.) dextrin, maltotriose, maltose and glucose that were detected by high performance liquid chromatography.