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1.
Determination of the respiration kinetics for mycelial pellets of Phanerochaete chrysosporium. 总被引:2,自引:2,他引:0
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In mycelial pellet cultures of the white rot basidiomycete Phanerochaete chrysosporium, low oxygen concentration negatively affects the production of the extracellular lignin peroxidases and manganese peroxidases which are key components of the lignin-degrading system of this organism. To test the hypothesis that oxygen limitation in the pellets is responsible for this effect, oxygen microelectrodes were used to determine oxygen concentration gradients within the mycelial pellets of P. chrysosporium. Pellets were removed from oxygenated cultures, allowed to equilibrate with air, and probed with oxygen microelectrodes. The oxygen profiles were modelled assuming that O2 uptake follows a Michaelis-Menten relationship. The Vmax and Km values for oxygen uptake were 0.76 +/- 0.10 g/m3 of pellet per s and 0.5 +/- 0.3 g/m3, respectively. These kinetic values were used to predict respiration rates in air-flushed cultures, oxygen-flushed cultures, and cultures with large pellets (diameter greater than 6 mm). The predicted respiration rates were independently validated by experimentally measuring the evolution of carbon dioxide from whole cultures. 相似文献
2.
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. 相似文献
3.
Modelling the growth kinetics of Phanerochaete chrysosporium in submerged static culture. 总被引:1,自引:0,他引:1
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The potential commercial application of Phanerochaete chrysosporium requires methods for quantitatively predicting growth and substrate utilization. The growth kinetics of P. chrysosporium INA-12 (CNCM I-398) were investigated and modelled under nonlimiting nitrogen and carbon conditions in submerged static culture. This strain, unlike other strains, does not require nutrient limitation for induction of lignin peroxidase. Maximum levels of lignin peroxidase activity were reached 7 days after culture initiation, when almost 80% of the initial glycerol and 70% of the initial nitrogen were still present. Lignin peroxidase levels then decreased, while biomass levels increased until about day 14. The ratio of cell dry weight to wet weight was constant until the maximum biomass concentration was achieved, after which there was a decrease in the water content. The change in this ratio reflects cell lysis as it correlated with increased concentrations of nitrogen in the media, arising from cell leakage. The suitability of four growth models to predict growth, and in some cases glycerol consumption, was evaluated. A simple linear model and the Emerson model performed poorly for the early stages of growth, while a modified Williams model and the Monod model predicted substrate and biomass concentrations equally well. All models will predict biomass concentrations during the active growth phase, but they should not be used to predict biomass concentrations after the stationary growth phase, when cell lysis becomes significant. 相似文献
4.
Wet Phanerochaete chrysosporium dried at 40°C yields about 3 times higher specific surface area (0.89 m2/g) than when dried at 80°C (0.34 m2/g) or 100°C (0.32 m2/g). An increased rate of evaporation and thermal expansion of water at the higher temperatures is probably responsible. The pellets dried at 40°C have approx. twice the Cu2 adsorption capacity (10.5 mg Cu2/g) than the pellets dried at 80°C (6.1 mg Cu2/g) or 100°C (5.8 mg Cu2/g). © Rapid Science Ltd. 1998 相似文献
5.
Metabolism of phenanthrene by Phanerochaete chrysosporium. 总被引:9,自引:0,他引:9
J B Sutherland A L Selby J P Freeman F E Evans C E Cerniglia 《Applied and environmental microbiology》1991,57(11):3310-3316
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. 相似文献
6.
J B Sutherland A L Selby J P Freeman F E Evans C E Cerniglia 《Applied microbiology》1991,57(11):3310-3316
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. 相似文献
7.
Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism 总被引:15,自引:0,他引:15
H Wariishi H B Dunford I D MacDonald M H Gold 《The Journal of biological chemistry》1989,264(6):3335-3340
Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme. 相似文献
8.
9.
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. 相似文献
10.
J A Bumpus 《Applied microbiology》1989,55(1):154-158
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. 相似文献
11.
Metabolism of cyanide by Phanerochaete chrysosporium 总被引:1,自引:0,他引:1
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. 相似文献
12.
Linko S 《Biotechnology advances》1992,10(2):191-236
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. 相似文献
13.
Diffuse reflectance spectrophotometry was applied for measuring ligninase activity in pellets of Phanerochaete chrysosporium. Enhanced ligninase activity in pellets and in growth medium were detected in cultures supplemented with oleic acid emulsified with Tween 80, Tween 80, hydrophilic (alcoholic) residue of Tween 80 hydrolysate, Tween 20, and (15OE)C18:1. In cultures with low extracellular ligninase activity, low activity in pellets was also observed. Our results indicate that the stimulatory effect of the tested surfactants cannot be contributed solely to promotion of ligninases through the cell membrane.
Correspondence to: D. Letan 相似文献
14.
Nine proteins with lignin peroxidase activity were separated from cultures of Phanerochaete chrysosporium INA-12 in glycerol as carbon source and non-nitrogen limited. Four lignin peroxidase isozymes (4, 5, 8, 9) were purified and characterized. Although differences in kinetic parameters could be shown, antibody reaction showed homology between isozymes. However, thermal stability studied, peptide mapping results, and N-terminal sequence analyses established a higher degree of homology between isozymes 4/5 and 8/9 types. Protein characterization and kinetic data indicate that lignin peroxidase isozymes 4, 5, 8, and 9 differ from described isozymes in strain BKM. The higher specific activity of lignin peroxidase isozymes in cultures with glycerol than in nitrogen-starved cultures accounts for the higher lignin peroxidase activity obtained in these conditions. 相似文献
15.
The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
Alternative methods for production and staining of Phanerochaete chrysosporium basidiospores.
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Homokaryotic isolates of Phanerochaete chrysosporium are generally obtained by stimulating the production of basidiospores. The most commonly used method requires a special incubator that is maintained at 28 degrees C with continuous illumination. Here we report an alternate method which permits the production of basidiopores with common laboratory incubators and requires no special illumination conditions. This alternate method gives reproducible results and yields basidiospores that are not contaminated with conidia. We also report a detailed optimized method for staining basidiospores for visualizing nuclei. 相似文献
17.
Electrophoretic karyotyping of the lignin-degrading basidiomycete Phanerochaete chrysosporium 总被引:1,自引:0,他引:1
Trevor M. D'Souza S. Balachandra Dass Avraham Rasooly C. Adinarayana Reddy 《Molecular microbiology》1993,8(5):803-807
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. 相似文献
18.
Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium. 总被引:5,自引:5,他引:5
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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). 相似文献
19.
Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium. 总被引:10,自引:6,他引:4
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Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (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 extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place. 相似文献
20.
A selective medium was developed that is capable of isolating Phanerochaete chrysosporium from soil. This medium contains 15 ppm of benomyl (15 μg g−1) and 550 ppm of streptomycin sulfate in 2% malt agar and is held at 39°C after inoculation. P. chrysosporium was isolated from three nonsterile forest soils to which the fungus had been added. These soils contained large microbial populations. 相似文献