Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

Abstract Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana . Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium . Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium , despite differences between brown and white rot modes of decay.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.     

Evidence for cytochrome P-450 and P-450-mediated benzo(a) pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium

Abstract The presence of cytochrome P-450 and P-450-mediated benzo(a)pyrene hydroxylase activity in both microsomal and soluble fractions of the white rot fungus Phanerochaete chrysosporium was shown. The reduced carbon monoxide difference spectrum showed maxima at 448–450 and 452–454 nm for microsomal and cytosolic fractions, respectively. Both P-450 fractions produced a Type I substrate binding spectrum on addition of benzo(a)pyrene. Activity for benzo(a)pyrene hydroxylation was NADPH-dependent and inhibited by carbon monoxide. K _m values for activity showed a difference between the cellular fractions with a K _m of 89 μM for microsomal P-450 and 400 μM for cytosolic P-450. The V _max values observed were 0.83 nmol min⁻ (nmol microsomal P-450) ⁻¹ and 0.4 nmol min⁻¹ (nmol cytosolic P-450)⁻¹. The results indicate that P-450-mediated benzo(a)pyrene hydroxylase activity could play a role in xenobiotic transformation by this fungus beside the known ligninolytic exocellular enzymes.     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Degradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium grown in ammonium lignosulphonate media

Removal and degradation of pentachlorophenol (PCP) by Phanerochaete chrysosporium in static flask cultures was studied using ammonium lignosulphonates (LS), a waste product of the papermill industry, as a carbon and nitrogen source. After 3 days, cultures of P. chrysosporium grown in either a 2% LS (nitrogen-sufficient) medium or a 0.23% LS and 2% glucose (nitrogen-deficient) medium removed 72 to 75% of PCP, slightly less than the 95% removal seen using nitrogen-deficient glucose and ammonia medium. PCP dehalogenation occurred despite the fact that extracellular enzyme (LiP) activity, measured by a veratryl alcohol oxidation assay and by PCP disappearance in cell-free extracts, was inhibited by LS. This inactivation of LiP likely contributed to the lower percent of PCP dehalogenation observed using the LS media. In order to better understand the relationship between PCP disappearance and dehalogenation, we measured the fate of the chlorine in PCP. After 13 days, only 1.8% of the initial PCP added was recoverable as PCP. The remainder of the PCP was either mineralized or transformed to breakdown intermediates collectively identified as organic halides. The largest fraction of the original chlorine (58%) was recovered as organic (non-PCP) halide, most of which (73%) was associated with the cell mass. Of the remaining chlorine, 40% was released as chloride ion, indicating a level of dehalogenation in agreement with previously reported values.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Biodegradation of bioaccessible textile azo dyes by Phanerochaete chrysosporium

Azo dyes are important chemical pollutants of industrial origin. Textile azo dyes with bioaccessible groups for lignin degrading fungi, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), were synthesised using different aminobenzoic and aminosulphonic acids as diazo components. The inocula of the best biodegradation assays were obtained from a pre-growth medium (PAM), containing one of the synthesised dyes. The results of the dye biodegradation assays were evaluated every 7 days, by the decrease of the absorbance at the maximum wavelength of the dye, by the decrease of the sucrose concentration in the culture medium and by the increase of the biomass during the 28 days of assay. It was observed that the extent of dye biodegradation depended on the sucrose concentration, on the degraded dye structure and, on the dye present in the PAM medium.     

黄孢原毛皮革菌降解苯胺的工艺研究

通过正交试验优化筛选了适合黄孢原毛皮革菌降解苯胺的适宜培养基和摇瓶培养降解条件。结果表明：其适宜降解的液体培养基组成为：蔗糖20g／L,可溶性淀粉20g／L,(NH4)2SO4l0g／L,Mn^2 lμmol／L,Tween-800．3％,蛋白胨30g／L。适宜降解的摇瓶培养条件为：接种量为20％、pH为7．0、温度为30℃、培养时间为12d．此条件下的苯胺最高降解率可达95．5％。     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium

Biodegradation of crystal violet (N,N,N',N',N',N'-hexamethylpararosaniline) in ligninolytic (nitrogen-limited) cultures of the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance of crystal violet and by the identification of three metabolites (N,N,N',N',N'-pentamethylpararosaniline, N,N,N',N'-tetramethylpararosaniline, and N,N',N'-trimethylpararosaniline) formed by sequential N-demethylation of the parent compound. Metabolite formation also occurred when crystal violet was incubated with the extracellular fluid obtained from ligninolytic cultures of this fungus, provided that an H2O2-generating system was supplied. This, as well as the fact that a purified ligninase catalyzed N-demethylation of crystal violet, demonstrated that biodegradation of crystal violet by this fungus is dependent, at least in part, upon its lignin-degrading system. In addition to crystal violet, six other triphenylmethane dyes (pararosaniline, cresol red, bromphenol blue, ethyl violet, malachite green, and brilliant green) were shown to be degraded by the lignin-degrading system of this fungus. An unexpected result was the finding that substantial degradation of crystal violet also occurred in nonligninolytic (nitrogen-sufficient) cultures of P. chrysosporium, suggesting that in addition to the lignin-degrading system, another mechanism exists in this fungus which is also able to degrade crystal violet.     

Non-Ligninolytic TNT Mineralization in Contaminated Soil by Phanerochaete chrysosporium

The explosive 2,4,6-trinitrotoluene (TNT) is widely used and results in widespread soil contamination. The white-rot fungus Phanerochaete chrysosporium has been shown to degrade TNT, using the peroxidase enzyme. In this study, we report peroxidase-independent degradation of TNT by non-ligninolytic P. chrysosporium. Significant disappearance of TNT from highly contaminated soil using P. chrysosporium has been observed. Soil highly contaminated with TNT (2270 ppm [10 mM]) was diluted to 100 ppm (0.44 mM) with malt extract medium. Pregrown (48 hours) mycelial pellets of P. chrysosporium were added in 100 mL malt extract medium and incubated in Gledhill flasks. Analysis by high-performance liquid chromatography (HPLC) was conducted on soil extracts at specific time points to estimate the disappearance of TNT from contaminated soil incubated with P. chrysosporium. When the pregrown mycelial pellets were added, TNT disappeared within 48 hours. The dissolved concentration of 2-amino-4,6-dinitrotoluene (2Am-DNT) increased up to the third day, then declined before its final disappearance by day 10. Results show that the pregrown mycelial pellets of P. chrysosporium mineralized up to 17.3±6.3% [¹⁴C]-TNT within 30 days.     

黄孢原毛平革菌生产锰过氧化物酶的发酵条件研究

目的 :研究黄孢原毛平革菌产锰过氧化物酶的发酵条件。方法 :对培养条件进行优化 ,采用正交设计法对培养基组分进行优化。结果与结论 :优化培养条件为 :接种量 1 6× 10 6 个孢子 L ,pH 4 4～ 4 8,温度 36℃～ 4 0℃ ,转数 12 0r/min。优化培养基参数为 :葡萄糖 5g L ,酒石酸铵 1 3mmol L ,吐温 - 80 1 2g L ,Mn2 + 0 9mmol L。     

Solid-state fermentation of natural birch lignin by Phanerochaete chrysosporium

A strain of white rot fungus, Phanerochaete chrysosporium Burds. ME446, has been characterized with respect to the extent and rate of Betula nigrificans lignin and non-lignin conversion by solid-substrate fermentation for different culture conditions. Moisture content, inoculum density, nitrogen supplementation and autoclaving of birch solids significantly affected lignin conversion rates and yields in 20 day fermentations. Oxygen favoured lignin over non-lignin conversion at partial pressures of 1.0 atm. Oxygen pressures of 2.0 atm severely inhibited both lignin and non-lignin conversions. Carbon dioxide partial pressures of 0.25, 0.5 and 1.0 atm at oxygen pressures of 1.0 atm increasingly inhibited both lignin and non-lignin conversion rates and yields. The results of these studies demonstrate the effects of major process variables and suggest a need to control the gas environment for process optimization.     

黄孢原毛平革菌产絮凝剂营养条件优化

微生物絮凝剂与传统化学絮凝剂相比,安全无毒、无二次污染,具有开发潜力.黄孢原毛平革菌(Phanerochaete chrysosporium)能产生微生物絮凝剂,但目前缺少对其产絮凝剂营养条件的优化.使用高岭土并利用单因素法研究碳源、氮源、碳氮比、接种量对Phanerochaete chrysosporium产絮凝剂的...     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium.

Biodegradation of crystal violet (N,N,N',N',N',N'-hexamethylpararosaniline) in ligninolytic (nitrogen-limited) cultures of the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance of crystal violet and by the identification of three metabolites (N,N,N',N',N'-pentamethylpararosaniline, N,N,N',N'-tetramethylpararosaniline, and N,N',N'-trimethylpararosaniline) formed by sequential N-demethylation of the parent compound. Metabolite formation also occurred when crystal violet was incubated with the extracellular fluid obtained from ligninolytic cultures of this fungus, provided that an H2O2-generating system was supplied. This, as well as the fact that a purified ligninase catalyzed N-demethylation of crystal violet, demonstrated that biodegradation of crystal violet by this fungus is dependent, at least in part, upon its lignin-degrading system. In addition to crystal violet, six other triphenylmethane dyes (pararosaniline, cresol red, bromphenol blue, ethyl violet, malachite green, and brilliant green) were shown to be degraded by the lignin-degrading system of this fungus. An unexpected result was the finding that substantial degradation of crystal violet also occurred in nonligninolytic (nitrogen-sufficient) cultures of P. chrysosporium, suggesting that in addition to the lignin-degrading system, another mechanism exists in this fungus which is also able to degrade crystal violet.     

Diuron degradation by Phanerochaete chrysosporium BKM- F-1767 in synthetic and natural media

When incubated in synthetic (N-limited) medium and on ashwood chips, Phanerochaete chrysosporium BKM-F-1767 degraded 14 and 10 mg/l diuron, respectively. The wood chips were used as support and sole nutrient source for the fungus. A higher degradation efficiency was found in ashwood culture as compared to the liquid culture, probably as a result of the synergetic effect of attached fungal growth, presence of limiting-substrate conditions and the microenvironment provided by ashwood, all favorable for production of high extracellular enzyme titres. Diuron degradation occured during the idiophasic growth, in the presence of manganese peroxidase, detected as dominant enzyme in both cultures.