Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

The Phanerochaete chrysosporium secretome: database predictions and initial mass spectrometry peptide identifications in cellulose-grown medium

The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome predictions and to evaluate the usefulness of the current database, we performed shotgun LC-MS/MS on cultures grown on standard cellulose-containing medium. A total of 182 unique peptide sequences were matched to 50 specific genes, of which 24 were among the secretome subset. Underscoring the rich genetic diversity of P. chrysosporium, identifications included 32 glycosyl hydrolases. Functionally interconnected enzyme groups were recognized. For example, the multiple endoglucanases and processive exocellobiohydrolases observed quite probably attack cellulose in a synergistic manner. In addition, a hemicellulolytic system included endoxylanases, alpha-galactosidase, acetyl xylan esterase, and alpha-l-arabinofuranosidase. Glucose and cellobiose metabolism likely involves cellobiose dehydrogenase, glucose oxidase, and various inverting glycoside hydrolases, all perhaps enhanced by an epimerase. To evaluate the completeness of the current database, mass spectroscopy analysis was performed on a larger and more inclusive dataset containing all possible ORFs. This allowed identification of a previously undetected hypothetical protein and a putative acid phosphatase. The expression of several genes was supported by RT-PCR amplification of their cDNAs.     

Xylanase Activity of Phanerochaete chrysosporium

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30°C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-β-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. β-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. β-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

Production of Phanerochaete chrysosporium lignin peroxidase

Liginin peroxidase (ligninase) of the white rot fungus Phanerochaete chrysosporium Burdsall was discovered in 1982 as a secondary metabolite. Today multiple isoenzymes are known, which are often collectively called as lignin peroxidase. Lignin peroxidase has been characterized as a veratryl alcohol oxidizing enzyme, but it is a relatively unspecific enzyme catalyzing a variety of reactions with hydrogen peroxide as the electron acceptor. P. chrysosporium ligninases are heme glycoproteins. At least a number of isoenzymes are also phosphorylated. Two of the major isoenzymes have been crystallized. Until recently lignin peroxidase could only be produced in low yields in very small scale stationary cultures owing to shear sensitivity. Most strains produce the enzyme only after grown under nitrogen or carbon limitation, although strains producing lignin peroxidase under nutrient sufficiency have also been isolated. Activities over 2000 U dm(-3) (as determined at 30 degrees to 37 degrees C) have been reported in small scale Erlenmeyer cultures with the strain INA-12 grown on glycerol in the presence of soybean phospholipids under nitrogen sufficiency. In about 8 dm(3) liquid volume pilot scale higher than 100 U dm(-3) (as determined at 23 degrees C) have been obtained under agitation with immobilized P. chrysosporium strains ATCC 24725 or TKK 20512. Good results have been obtained for example with nylon web, polyurethane foam, sintered glass or silicon tubing as the carrier. The immobilized biocatalyst systems have also made large scale repeated batch and semicontinuous production possible. With nylon web as the carrier, lignin peroxidase production has recently been scaled up to 800 dm(3) liquid volume semicontinuous industrial production process.     

Biocatalytic decolourisation of molasses by Phanerochaete chrysosporium

Bioremediation potential of Phanerochaete chrysosporium strains NCIM 1073, NCIM 1106 and NCIM 1197 to decolourise molasses in solid and liquid molasses media was studied. Strains varied in the pattern of molasses decolourisation on solid medium by Giant colony method. Under submerged cultivation conditions, strain NCIM 1073 did not decolourise molasses while, strains NCIM 1106 and NCIM 1197 could decolourise molasses up to 82% and 76%, respectively. Under stationary cultivation conditions, none of the strains could decolourise molasses. This was overcome by increasing the surface area of the culture in flat bottom glass bottles under stationary cultivation conditions. Under submerged cultivation conditions, growth was more or less same in all strains. However, the lignin peroxidase and manganese peroxidase activities were significantly less in the strain NCIM 1073. Under stationary cultivation conditions, none of the strains could produce enzymes lignin peroxidase, manganese peroxidase and laccase. However, all of them could produce lignin peroxidase and manganese peroxidase when cultivated in flat bottom glass bottles under stationary cultivation conditions.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.     

Kinetics of endosulfan degradation by Phanerochaete chrysosporium

The chlorinated pesticide, endosulfan, could be degraded by Phanerochaete chrysosporium under non-ligninolytic conditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfan diol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan l^–1, degradation was at 0.23 mg l^–1 day^–1. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.     

Biodegradation of Rose Bengal by Phanerochaete chrysosporium

Rose Bengal (tetrachloro-tetraiodo-fluorescein) was not able to limit the spreading growth of the ligninolytic fungus, Phanerochaete chrysosporium in the presence of Tween 80, and when added to the 5 d old liquid cultures of this organism it was almost completely degraded in 5 h. Thin layer chromatography analysis showed the formation of a single degradation product.     

Biodegradation of polycyclic hydrocarbons by Phanerochaete chrysosporium

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 methylene chloride and particulate fractions, respectively. High-performance liquid chromatography of the 14C-labeled material present in the methylene chloride fraction revealed that most (91.9%) of this material was composed of polar metabolites of [14C]phenanthrene. These results suggest that this microorganism may be useful for the decontamination of sites in the environment contaminated with PAHs.     

Semi-continuous ligninase production using foam-immobilised Phanerochaete chrysosporium

Summary The immobilisation of Phanerochaete chrysosporium in cubes of polyurethane foam enables ligninase to be produced on a semi-continuous basis. At each successive harvest, cultures are purged with oxygen and ligninase activity induced with veratryl alcohol. Using 200 ml of a five-fold dilution of the batch culture medium in 1l flasks, harvests of ligninase with the same apparent protein profile by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are obtained every 24 to 48 h.It is also possible to store cubes of foam containing pellets of P. chrysosporium so that further yields of ligninase comparable to fresh cultures can be produced within 48 h when desired.     

Degradation kinetics of pentachlorophenol by Phanerochaete chrysosporium

The extracellular enzymes and cell mass from the pregrown Phanerochaete chrysosporium cultures were used for the degradation of PCP. The use of both extracellular enzymes and cell mass resulted in extensive mineralization of PCP, while the action of only the crude extracellular enzymes led to the formation of a degradation intermediate (TCHD). A kinetic model, which describes the relationship among PCP degradation, initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration, was developed. Based on this model, various effects of initial PCP concentration, dosage of extracellular enzymes, and cell mass concentration were evaluated experimentally. It was found that when initial PCP concentration is lower than 12 mumol/L, the model of a parallel-series first-order reaction is sufficient to describe the degradation process. PCP disappearance and mineralization were enhanced by increasing either the extracellular enzyme concentration or the cell mass concentration. As high as 70% of PCP mineralization could be obtained by using a higher dosage of extracellular enzymes and cell mass. Various parameters of the kinetic model were determined and the model was verified experimentally. Simulation using this model provided the criteria needed to choose rational dosages of extracellular enzymes and cell mass for the degradation of PCP. Data reported allow some insight into the function of the extracellular enzymes and cell mass of P. chrysosporium in degradation processes of toxic pollutants and assist in the design and evaluation of practical bioremediation methods.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium

Electrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF-1767 and ME-446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF-1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME-446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF-1767 and to four chromosomes in strain ME-446.     

Nutritional Regulation of Lignin Degradation by Phanerochaete chrysosporium

Previous studies have shown that a lignin-degrading system appears in cultures of the white rot fungus Phanerochaete chrysosporium in response to nitrogen starvation, apparently as part of secondary metabolism. We examined the influence of limiting carbohydrate, sulfur, or phosphorus and the effect of varying the concentrations of four trace metals, Ca, and Mg. Limitation of carbohydrate or sulfur but not limitation of phosphorus triggered ligninolytic activity. When only carbohydrate was limiting, supplementary carbohydrate caused a transient repression of activity. In carbohydrate-limited cultures, ligninolytic activity appeared when the supplied carbohydrate was depleted, and this activity was associated with a decrease in mycelial dry weight. The amount of lignin degraded depended on the amount of carbohydrate provided, which determined the amount of mycelium produced during primary growth. Carbohydrate-limited cultures synthesized only small amounts of the secondary metabolite veratryl alcohol compared with nitrogen-limited cultures. l-Glutamate sharply repressed ligninolytic activity in carbohydrate-starved cultures, but NH(4) did not. Ligninolytic activity was also triggered in cultures supplied with 37 muM sulfur as the only limiting nutrient. The balance of trace metals, Mg, and Ca was important for lignin degradation.     

Bacteria Are Omnipresent on Phanerochaete chrysosporium Burdsall

Bacteria have been isolated from 10 different strains of Phanerochaete chrysosporium, a white rot fungus which degrades lignocellulosic materials. The investigations showed that one or more bacterial species were always associated with the fungus. Various attempts to eliminate the bacteria on the fungus were unsuccessful. Three different bacterial species were isolated and identified. One of these was Agrobacterium radiobacter, while another may represent a new taxon close to the genus Burkholderia. A third strain remains unidentified but is most probably a member of rRNA superfamily IV or the Woese (alpha) group. Besides P. chrysosporium, 23 other white rot fungi and 9 brown rot fungi were also investigated. None of these was associated with bacteria. The physiological significance of the association between the fungus and the bacteria remains to be elucidated.