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1.
The production of ligninolytic enzymes was studied in surface cultures of the South American white-rot fungus Nematoloma frowardii b19 and four other strains of this ecophysiological group (Clitocybula dusenii b11, Auricularia sp. m37a, wood isolates u39 and u45), which are able to depolymerize low-rank-coal-derived humic acids with the formation of fulvic-acid-like compounds. The fungi produced the three crucial enzymes of lignin degradation – lignin peroxidase, manganese peroxidase and laccase. In the case of N. frowardii b19, laccase and the two peroxidases could be stimulated by veratryl alcohol. Manganese (II) ions (Mn2+) caused a rapid increase of Mn peroxidase activity accompanied by the complete repression of lignin peroxidase. Under nitrogen-limited conditions the growth as well as the production of ligninolytic enzymes was partly repressed. During the depolymerization process of coal humic acids using solid agar media, gradients of ligninolytic enzyme activities toward 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate) and syringaldazine were detectable inside the agar medium. Received: 5 August 1996 / Received revision: 13 November 1996 / Accepted: 15 November 1996  相似文献   

2.
White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood. This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure. Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes. In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants. Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes. It is known that supplementing the growth medium with either Mn2+, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities. Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63–2. Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added. However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase. Received: 4 February 2000 / Received revision: 11 May 2000 / Accepted: 12 May 2000  相似文献   

3.
The majority of lignin-degrading basidiomycetes are able to depolymerize humic acids. In this presentation the relationship and possible similarities between enzymes involved in lignin degradation and humic acid depolymerization were examined on the genetic level. We have cloned fragments of the gene encoding the extracellular ligninolytic enzyme laccase from Clitocybula dusenii, Nematoloma frowardii and a fungal strain designated i63-2, and compared the three sequences with those of several other published laccase genes. The sequenced fragments displayed a high homology both on the DNA (97%–77%) and amino acid (100%–85%) level. Furthermore, the expression of this gene in the above-mentioned fungi was demonstrated by a nested polymerase chain reaction with cDNA as template. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998  相似文献   

4.
Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l−1 phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn2+ and NaN3 were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.  相似文献   

5.
Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation. Received: 30 March 2000 / Accepted: 19 May 2000  相似文献   

6.
Production of ligninolytic enzymes and degradation of 14C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for 14C dehydrogenative polymerizates (DHP; 38% 14CO2 after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% 14CO2 after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of 14C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes. Received: 29 January 1999 / Received revision: 5 July 1999 / Accepted: 9 July 1999  相似文献   

7.
d-Xylose/d-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these d-xylose isomerases are homotetramers with relative subunit molecular masses of 45 000 and 53 000, respectively, as estimated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the d-xylose isomerase from Paenibacillus sp. has the highest activity with Mn2+, while the enzyme from A. ruhlandii prefers Mg2+. Both enzymes also accept Co2+ with a somewhat lower efficiency, while Cu2+ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identical binding sites per subunit. d-Glucose is converted to d-fructose at a rate that is two- to three-fold slower than for the d-xylose isomerisation. d-Xylitol and d-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active up to 60 °C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 °C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 °C. Polyacrylamide entrapment and immobilisation to both controlled pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency. Received: 14 May 1998 / Received last revision: 29 July 1998 / Accepted: 29 July 1998  相似文献   

8.
The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn2+. Mn2+ was especially related to the highest substrate consumption. Aerobiosis and Mn2+ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H2O2 meant that the survival of air-grown cells supplied with Mn2+ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn2+ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H2O2 treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1. Received: 24 July 1998 / Received last revision: 11 November 1998 / Accepted: 13 November 1998  相似文献   

9.
Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic 14C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%–50% of the initially added radioactivity was released as 14CO2). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days. Received: 31 July 1998 / Received revision: 29 September 1998 / Accepted: 2 October 1998  相似文献   

10.
Manganese peroxidase, MnP, is one of the major ligninolytic enzymes produced by a number of white-rot fungi. The ability of this enzyme to degrade lignin by the fungus Bjerkanderasp BOS55 has opened its application to related bioprocesses such as recalcitrant-compound degradation and effluent decolorization. The medium reported to induce MnP production is composed of chemical grade reagents, all with relatively high costs for application to detoxification purposes. The use of inexpensive sources for MnP production can bring its implementation closer. For this purpose, dairy residues from cheese processing were considered. MnP production obtained using crude whey as the sole substrate reached appreciable levels, around 190 U L−1, values comparable to those found with synthetic media (between 175–250 U L−1). Thus, this cheese-processing byproduct can be used as an inexpensive alternative for the large-scale production of MnP. Received 14 December 1998/ Accepted in revised form 29 April 1999  相似文献   

11.
Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn2+, Ca2+, Mg2+, and Sr2+ ions in the reaction mixture, and its thermal stability was significantly increased by Ca2+ ion. Received: 31 August 1998 / Received last revision: 1 December 1998 / Accepted: 20 December 1998  相似文献   

12.
The agaric basidiomycete Clitocybula dusenii was used for the production of the extracellular ligninolytic enzyme, manganese (Mn) peroxidase. An immobilization technique is described using cellulose and polypropylene as carrier for the fungal mycelium. High amounts of Mn peroxidase were obtained with agitated cultures of immobilized fungus (up to 3,000 U l−1) while the biomass was recovered and used for further production cycles. Purification of Mn peroxidase revealed the existence of two forms: MnP1 (molecular mass 43 kDa, pI 4.5) and MnP2 (42 kDa, pI 3.8). Received: 30 July 1999 / Received revision: 1 December 1999 / Accepted: 3 December 1999  相似文献   

13.
The frequency of occurrence of the litter-decomposing basidiomycetes ofPicea abies andP. glehnii forest floors in Hokkaido was investigated. In both theP. abies andP. glehnii forest plots (each 10 m×10 m), litter-decomposing basidiomycetes of the generaCollybia andMycena were frequently observed. Species composition, frequency of occurrence, and basidioma numbers of each species were different between the two forest plots, but several species were common to both. Seven species isolated from theP. glehnii forest plot (C. acervata, C. pinastris, Marasmius pallidocephalus, Ma. wettsteinii, My. aurantiidisca, My. clavicularis, Mycena sp. 1) and four species from theP. abies forest plot (C. pinastris, My. alphitophora (=My. osmundicola), Mycena sp. 1,My. vulgaris) were tested for their ability to degrade lignin by a simple plate test for extracellular phenoloxidases and by measuring Klason lignin loss from needle litter of spruce. All the strains of the litter-decomposing fungi tested showed positive reactions on the plate test. Lignin contained in the needle litter was degraded by all strains tested (onlyMy. alphitophora was not tested), and rates varied from 9% to 40% over a two-month period in vitro. Seven species with ligninolytic ability in theP. glehnii forest plot and four such species in theP. abies forest plot were found respectively in 77% and 60% of the 100 subplots in each plot. The results of this study suggest that lignin decomposition of needle litter by litter-decomposing basidiomycetes in the forest floor is a common event in the studiedPicea forests of Hokkaido and that the diversity of the ligninolytic activity among the species or strains may cause spatial heterogeneity of litter decomposition in thePicea forest floor.  相似文献   

14.
Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene   总被引:6,自引:0,他引:6  
Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [14C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [14C]TNT (100 μM corresponding to 4.75 μCi/l) to 14CO2 within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [14C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996  相似文献   

15.
Degradation of 2,4,6-trinitrotoluene (TNT) by the white-rot fungus Bjerkandera adusta DSM 3375 was studied in relation to extracellular ligninolytic activities. The Mn(II)-dependent peroxidase, the only ligninolytic enzyme detectable, reached a maximum activity of 600 ± 159 U/l after incubation in mineral medium with a sufficient nitrogen source. In contrast, the highest extent of [14C]TNT mineralization was detected in malt extract broth, so that the ability of B. adusta to mineralize TNT did not parallel ligninolytic activity. The microsomal fraction of cells grown in the presence of TNT was found to contain 11 pmol cytochrome P-450/mg protein. In cells grown without TNT, no microsomal cytochrome P-450 could be found. Instead, 14 pmol P-450/mg protein was present in the cytosolic fraction of these cells. Cytochrome P-450 apparently affected the TNT metabolism, as shown by inhibitory studies. Addition of the cytochrome P-450 inhibitor piperonyl butoxide diminished the 14CO2 release from 21% to 0.9%, as determined after 23 days of incubation, while 1-aminobenzotriazole and metyrapone decreased the mineralization to 8.6% and 6.3% respectively. Mass-balance analysis of TNT degradation in liquid cultures revealed that, by inhibition of cytochrome P-450, the TNT-derived radioactivity associated with biomass and with polar, water-soluble metabolites decreased from 93.9% to 15.0% and the fraction of radiolabelled metabolites extractable with organic solvents fell to 92.6%. The TNT metabolites of this fraction were identified as aminodinitrotoluenes, indicating that this initial transformation product of TNT may function as a substrate for cytochrome-P-450-dependent reactions in B. adusta. Received: 27 May 1999 / Received revision: 19 August 1999 / Accepted: 19 August 1999  相似文献   

16.
Fraser PD  Schuch W  Bramley PM 《Planta》2000,211(3):361-369
 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K m values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000  相似文献   

17.
Inhibition of the growth of Saccharomyces cerevisiae was evident at concentrations of 0.5 mM Mn2+ or higher, but a tolerance to lower Mn2+ concentrations was observed. The inhibitory effects of 2.0 mM Mn2+ were eliminated by supplementing the medium with excess Mg2+ (10 mM), whereas addition of excess Ca2+ and K+ had negligible effect on Mn2+ toxicity. Growth inhibition by Mn2+, in the absence of a Mg2+ supplement, was attributed to Mn2+ accumulation to toxic intracellular levels. Mn levels in S. cerevisiae grown in Mg2+-supplemented medium were severalfold lower than those of cells growing in unsupplemented medium. Mn2+ toxicity was also influenced by intracellular Mg, as Mn2+ toxicity was found to be more closely correlated with the cellular Mg:Mn ratio than with cellular Mn levels alone. Cells with low intracellular levels of Mg were more susceptible to Mn2+ toxicity than cells with high cellular Mg, even when sequestered Mn2+ levels were similar. A critical Mg:Mn ratio of 2.0 was identified below which Mn2+ toxicity became acute. The results demonstrate the importance of intracellular and extracellular competitive interactions in determining the toxicity of Mn2+. Received: 18 June 1997 / Received last revision: 10 January 1998 / Accepted: 24 January 1998  相似文献   

18.
The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000  相似文献   

19.
The magnesium content of Saccharomyces cerevisiae was found to vary by up to fivefold at differing␣ stages of batch growth and during growth in the presence of differing magnesium concentrations. Excess Mg was primarily sequestered in vacuoles. Mn2+-uptake experiments revealed that Mg-enriched cells had a markedly reduced capacity for Mn2+ accumulation. For example, after 6 h incubation in the presence of 50 μM Mn2+, Mn levels were approximately twofold higher in cells previously grown in unsupplemented medium than in those from Mg-supplemented medium. These differences were further accentuated at higher Mn2+ concentrations and were not attributable to altered cell-surface charge or altered cell-surface Mn2+ binding. Cellular Mg status also influenced Mn toxicity towards S. cerevisiae. During exposure to 5 mM Mn2+, 50% reductions in the viability of cells with initial Mg contents of approximately 1400 and 2700 nmol (109 cells)−1 occurred after approximately 1.6 h and 3.6 h respectively. In cells containing 3300 nmol Mg (109 cells)−1, more than 75% viability was still maintained after 7 h incubation with 5 mM Mn2+. It is concluded that Mn2+ uptake and toxicity in S. cerevisiae are strongly influenced by intracellular Mg, possibly through Mg-dependent regulation of divalent-cation transport activity. Received: 15 May 1996 / Received revision: 13 September 1996 / Accepted: 22 September 1996  相似文献   

20.
Laccase, aryl-alcohol oxidase and superoxide radicals were detected in ligninolytic cultures of Fusarium proliferatum. Enzyme activities were present during the secondary metabolism and seen as single protein bands after non-denaturing electrophoresis. In contrast, superoxide radicals were detected during primary growth, correlating with maximal lignin mineralization. Moreover, ligninolysis decreased when scavengers of both superoxide and hydroxyl radicals were added to cultures, indicating that activated oxygen species are involved in lignin degradation. Received: 12 October 1998 / Accepted: 24 October 1998  相似文献   

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