Ligninolytic Enzyme Activities Research Articles

Atmospheric nitrate deposition and the microbial degradation of cellobiose and vanillin in a northern hardwood forest

Human activity has increased the amount of N entering terrestrial ecosystems from atmospheric NO 3 − deposition. High levels of inorganic N are known to suppress the expression of phenol oxidase, an important lignin-degrading enzyme produced by white-rot fungi. We hypothesized that chronic NO 3 − additions would decrease the flow of C through the heterotrophic soil food web by inhibiting phenol oxidase and the depolymerization of lignocellulose. This would likely reduce the availability of C from lignocellulose for metabolism by the microbial community. We tested this hypothesis in a mature northern hardwood forest in northern Michigan, which has received experimental atmospheric N deposition (30 kg NO 3 −–N ha −1 y −1) for nine years. In a laboratory study, we amended soils with 13C-labeled vanillin, a monophenolic product of lignin depolymerization, and 13C-labeled cellobiose, a disaccharide product of cellulose degradation. We then traced the flow of 13C through the microbial community and into soil organic carbon (SOC), dissolved organic carbon (DOC), and microbial respiration. We simultaneously measured the activity of enzymes responsible for lignin (phenol oxidase and peroxidase) and cellobiose (β-glucosidase) degradation. Nitrogen deposition reduced phenol oxidase activity by 83% and peroxidase activity by 74% when compared to control soils. In addition, soil C increased by 76%, whereas microbial biomass decreased by 68% in NO 3 − amended soils. 13C cellobiose in bacterial or fungal PLFAs was unaffected by NO 3 − deposition; however, the incorporation of 13C vanillin in fungal PLFAs extracted from NO 3 − amended soil was 82% higher than in the control treatment. The recovery of 13C vanillin and 13C cellobiose in SOC, DOC, microbial biomass, and respiration was not different between control and NO 3 − amended treatments. Chronic NO 3 − deposition has stemmed the flow of C through the heterotrophic soil food web by inhibiting the activity of ligninolytic enzymes, but it increased the assimilation of vanillin into fungal PLFAs.

Pre-screening of fungi and bulking agents for contaminated soil bioremediation

This paper discusses methods to promote fungal growth and penetration in a soil contaminated with weathered crude oil. The ligninolytic enzyme activity and toxicity threshold of several white-rot fungi known for their hydrocarbon degradation ability was studied. Pine wood chips, peat moss and Kellogg's Bran Flakes were examined for their properties as the bulking agents and solid amendments. The results showed all strains developed severe toxicity at concentrations higher than 10 000 ppm. Two strains of Bjerkandera adusta UAMH 7308 and 8258 showed the highest ligninolytic enzyme activity. Furthermore, white-rot fungi did not colonize the soil without bulking agents being present in the soil. A mixture of peat moss with bran flakes resulted in the best growth, penetration and enzyme activity in the soil.

Laccase and other ligninolytic enzyme activities of selected strains of Trametes spp. from different localities and substrates.

Eighty-three strains belonging to three species of the genus Trametes FR. (T. versicolor, T. hirsuta and T. ochracea) collected in different localities and on different substrates were screened for laccase production. The production of other lignin-modifying enzymes--manganese peroxidase (MnP) and lignin peroxidase (LiP)--and the decolorization ability were also determined in 21 of them. Production variability was relatively high and no significant correlation was found between the origin of the strains (locality, substrate) and the enzyme production. Dikaryons of all 3 species (but not of all their strains) exhibited LiP activity, which was not detected in the respective monokaryons.

Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium

Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-β-glucanase, exo-1,4-β-glucanase, 1,4-β-glucosidase, endo-1,4-β-xylanase, 1,4-β-xylosidase and 1,4-β-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-β-glucanase, 1,4-β-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-β-mannosidase in P. ostreatus.

Microsomal transformation of organophosphorus pesticides by white rot fungi.

The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot fungi strains was studied. With the exception of Ganoderma applanatum 8168, all strains from a collection of 17 different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These strains depleted 50 to 96% of terbufos, azinphos-methyl, phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the transformation activity, the extracellular and microsomal fractions of Pleuronts ostreatus 7989 were evaluated in vitro. While the activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found, suggesting the intracellular origin of the transformation activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with the following rates: 10 micromol mg prot(-1) h(-1) for phosmet, 5.7 micromol mg prot(-1) h(-1) for terbufos, and 2.2 micromol mg prot(-1) h(-1) for azinphos-methyl. The products from these reactions and from the transformation of trichlorfon and malathion, were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent requirement for NADPH during the in vitro assays suggest the involvement of a cytochrome P450.

Monochlorophenol의 목질분해균에 의한 분해 특성

Biodegradation of monochlorophenols by wood rot fungi such as Daldina concentrica, Trametes versicolor and Pleurotus ostreatus was evaluated by determining their resistance or toxic test and biodegradability. The metabolites of monochlorophenols were also analyzed. Among the three fungi, T. versicolor was the most resistant to 200 ppm of 2-, 3- and 4-chlorophenols, and did not show any inhibitory mycellium growth. But D. concentrica had a little inhibition effect at more than 100 ppm of 3- or 4-chlorophenol. Control cultures of P. ostreatus took even 14 days far the completion of mycellium growth, but the hyphal growth was improved when 2- or 3-chlorophenol were added to the culture. In biodegradation analysis, P. ostreatus showed the highest degradation of 2- or 3-chlorophenol, while T. versicolor was the most effective in 4-chlorophenol. D. concentrica and P. ostreatus slowly degraded 4-chlorophenol. However, T. versicolor had similar degradation capability in the three monochlorophenols, suggesting that the biode- gradation nude is dependent on the fungi as well as the type of monochlorophenol. Several metabolites such as 1,3,5-trihydroxyl benzene, 1-ethyl-1-hydroxyl pentane, 2-propenoicacid, methylmalonic acid and 2-methyl-4-keto-pentan-2-ol were found as products of primary oxidation of 2-, 3- and 4-chlorophenols by intact fungal cultures. fatty acids including tetradecanoic, heptadecanoic and octadecanoic acids were also detected The order of increase of mycellium weight during incubation were P. ostreatus > T. versicolor > D. concentrica. The pH in the culture was not constantly changed depending on incubation days, but the mycellium weight was slightly increased, indicating that the biodegradation of monochlorophenol might have low relationship with the mycellium growth Laccase activities of T. versicolor and P. ostreatus were continuously increased depending on the incubation days, suggesting that the ligninolytic enzyme activity play an important role in the biodegradation of monochlorophenol.

Performance of a solid-state immersion bioreactor for ligninolytic enzyme production: evaluation of different operational variables

The production of ligninolytic enzymes by the white-rot fungus Phanerochaete chrysosporium BKM-F-1767 in a solid-state immersion bioreactor, employing cubes of nylon sponge as a support, was studied. Cultivation was carried out in both batch and continuous mode, and the effect of some operational variables (aeration level, pH) was investigated. Batch operation at an aeration level of 0.5 vvm led to maximum manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 574 and 116 U l −1, respectively. The results were compared with those obtained at a higher aeration level (1 vvm), reported in a previous work, and it appeared that LiP productivity increased with aeration rate, while MnP was not significantly affected. Continuous operation showed much lower MnP activities (around 60 U l −1) and similar LiP activities (about 132 U l −1). However, in terms of productivity the difference between batch and continuous operation for MnP was less remarkable (239 and 150 U day −1, respectively), whereas LiP productivity was ten-fold higher in continuous operation than in batch mode. This could be attributed to the influence of operation pH on ligninolytic enzyme activities. The study of the kinetic characteristics of the biocatalysts supported this hypothesis.

Removal of surfactant solubilized polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium in a rotating biological contactor reactor

White rot fungi can oxidize surfactant solubilized polycyclic aromatic hydrocarbons (PAH). The objective of this study was to evaluate the performance of immobilized white rot fungus, Phanerochaete chrysosporium, to remove surfactant Tween 80 solubilized PAH i.e. phenanthrene, pyrene and benzo(α)pyrene in a rotating biological contactor (RBC) reactor. Results indicated that the immobilized P. chrysosporium in the RBC reactor system in continuous operation could effectively remove the three tested PAH at specific hydraulic loading rates and concentrations tested for each individual PAH. Batch operation of RBC reactor showed that the immobilized P. chrysosporium was stable and effective for the eight successive batch treatments of PAH in solution medium i.e. PAH removal was greater than 90% after 60 h, although only low levels of ligninolytic enzyme activity were detected. The removal of phenanthrene and pyrene in solution medium has been found to be a first order reaction in batch operation. A mass balance calculation indicated that biological oxidation was the main factor for removal of benzo(α)pyrene i.e. 95.7% in the RBC reactor. However, for phenanthrene and pyrene, both biological oxidation (i.e. 49 and 56%, respectively) and RBC disc foam adsorption (i.e. 44 and 34%, respectively) made a significant contribution to the removal of PAH.

木材腐朽菌によるケナフパルプ，シラカバパルプの漂白

White rot fungi OM 5-1 and KUF 2 isolated from the experimental forest of Kochi University had high abilities to brighten the kenaf bark pulp and the shirakaba pulp, respectively. Bleachabilities of both fungi were compared with those of standard fungi, i. e. Phanerochaete chrysosporium and Pleurotus ostreatus. Brightness increase of kenaf bark pulp by OM 5-1 was much larger than that estimated from kappa number decrease. The similar tendency was observed when the shirakaba kraft pulp was bleached by KUF 2. Bleachability of kenaf core pulp by fungi was not similar to that of kenaf bark pulp, but to that of shirakaba pulp.Cumulative activities of ligninolytic enzymes from each of above four fungi had linear relation to the pulp brightness increase obtained by each fungus treatment. But, during later periods of treatment only the kenaf bark pulp showed higher brightness out of this linear relationship. For kenaf bark pulp and shirakaba pulp, OM 5-1 and KUF 2 showed the highest pulp viscosities, respectively.

Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media

Solid state cultures of two white-rot fungi, Phanerochaete chrysosporium and Phlebia radiata, have been carried out, using an inert support (nylon sponge) and a support-substrate (corncob). The suitable medium and culture conditions have been chosen to favour the secretion of ligninolytic enzymes. The production of manganese peroxidase, lignin peroxidase, laccase and proteases has been monitored during the cultures, in an attempt to investigate the possible effect of the latter on the integrity of ligninolytic enzymes. The higher the protease concentration in the culture medium, the more irregular the profiles of ligninolytic enzyme activity. P. chrysosporium secretes proteolytic enzymes mainly during primary metabolism, while P. radiata produced these at the onset of secondary metabolism. Furthermore, different types of proteases produced were identified, P. chrysosporium secreted mainly thiol and acidic proteases, while P. radiata cultures contained thiol-, serin- and metalloproteases.

Strategies for improving ligninolytic enzyme activities in semi-solid-state bioreactors

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) was studied in laboratory-scale bioreactors, operating under semi-solid-state conditions. The bioreactors were filled with inert support (polypropylene sponges) and cultivation was carried out in batch. Veratryl alcohol (2 mM) and Tween 80 (0.05% v/v) were added at the beginning of the fermentation to stimulate the production of ligninolytic enzymes. A modification in the design of a bioreactor, which operates in semi-solid-state conditions, lead to a significant improvement in the ligninolytic enzyme activities obtained. Thus, by modifying the design of the bioreactor employed in the current study, it was possible to increase about tenfold manganese-dependent peroxidase (MnP) activities and around 40% lignin peroxidase (LiP) activities. Moreover, both MnP and LiP profiles were more regular in the modified bioreactor.

Effect of non‐ionic surfactants on elimination of polycyclic aromatic hydrocarbons (PAHs) in soil‐slurry by Phanerochaete chrysosporium

AbstractSurfactants can enhance bioremediation of soil contaminated with polycyclic aromatic hydrocarbons (PAHs) by increasing their bioavailability. The objective of this study was to evaluate the effect of non‐ionic polyoxyethylene (POE) surfactants on the elimination of nine PAHs in contaminated soil by the white rot fungus Phanerochaete chrysosporium. PAHs in both liquid and solid phases were extracted and then assayed using GC–MS. Fungal biomass concentration and the activity of extracellular ligninolytic enzymes were used to determine the toxicity of POE surfactants. Results indicated that 0.5% (w/v) of surfactant Tween 80 was not toxic to fungal growth and had little impact on extracellular ligninolytic enzyme synthesis. About 80% of low ring PAHs (ie acenaphthene and fluorene) could be efficiently catabolized by P chrysosporium without surfactants, while catabolism of others (ie phenanthrene, fluoranthene, pyrene, chrysene, benzo(a)pyrene, dibenz(ah)anthracene and benzo(ghi)perylene) was less than 30% over a 8‐day culture period. Elimination of four‐to six‐ring PAHs in contaminated soil was enhanced in P chrysosporium cultures with 0.4% (w/v) Tween 80 by 15–33% compared with those in cultures without Tween 80, while no obvious effect was observed in the elimination of three‐ring PAHs over the culture period. Investigations on PAH concentrations in aqueous phase during the culture period showed that Tween 80 increased PAH aqueous concentrations and the PAH oxidation rate in aqueous was rapid. Enhancement of soil PAH elimination in the presence of Tween 80 was due to the increased PAH bioavailability.© 2001 Society of Chemical Industry

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation.

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.

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

MICROBIAL ENZYME SHIFTS EXPLAIN LITTER DECAY RESPONSES TO SIMULATED NITROGEN DEPOSITION

Some natural ecosystems near industrialized and agricultural areas receive atmospheric nitrogen inputs that are an order of magnitude greater than those presumed for preindustrial times. Because nitrogen (N) often limits microbial growth on dead vegetation, increased N input can be expected to affect the ecosystem process of decomposition. We found that extracellular enzyme responses of a forest-floor microbial community to chronically applied aqueous NH4NO3 can explain both increased and decreased litter decomposition rates caused by added N. Microbes responded to N by increasing cellulase activity in decaying leaf litter of flowering dogwood, red maple, and red oak, but in high-lignin oak litter, the activity of lignin-degrading phenol oxidase declined substantially. We believe this is the first report of reduced ligninolytic enzyme activity caused by chronic N addition in an ecosystem. This result provides evidence that ligninolytic enzyme suppression can be an important mechanism explaining decreased ...

Influence of cadmium and mercury on activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil.

The white-rot fungus Pleurotus ostreatus was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[a]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.

Studies on the role of proteases in the white-rot fungusTrametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity

In the present study we investigated the possibility of proteinases, intracellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. These studies were performed using specific inhibitors added to the cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of serine proteinases) or chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) revealed distinct differences in the activity of ligninolytic enzymes between nutrient-deprived and non-starved cultures. The addition of PMSF during the transfer of mycelia to the nutrient limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxidase decreased with PMSF, both in tropho- and in idiophasic cultures. The enhanced activities of laccase and general peroxidases (horseradish peroxidase-like, HRP-like) were accompanied by markedly altered patterns of both intracellular and extracellular proteolytic activities revealed by electrophoretic analysis with polyacrylamide gels containing the copolymerized substrate (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar proteolysis resulted in lowered activities of laccase and peroxidase. Electrophoretic analysis revealed altered patterns of intracellular proteinases upon chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon-starved cultures. From the results it is concluded that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation.

Studies on the role of proteases in the white-rot fungus Trametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity

Studies on the role of proteases in the white-rot fungus Trametes versicolor: Effect of PMSF and chloroquine on ligninolytic enzymes activity

Screening of Pleurotus ostreatus isolates for their ligninolytic properties during cultivation on natural substrates.

Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derived isolates growing well in nonsterile soil were found, which could be suitable for the prospective biotechnological exploitation.