Mn Peroxidase Research Articles

Influence of Organic Acids on Kinetic Properties of Engineered <i>Phlebia radiata</i> MnP3 Enzymes (Wild-type and Mutants) during Mn (II) Oxidation

Organic acids are potent inhibitors or activators of enzymes and are involved in the catalytic cycle of peroxidase. This study investigated the influence of some organic acids on kinetic parameters during the oxidation of Mn (II) catalyzed by recombinant Phlebia radiata wild-type and mutant manganese peroxidases. For the wild-type, the highest affinity (low Km) and highest catalytic efficiency were obtained in the presence of lactate. The E40H, E44H, D186H and D186N rPr-MnP3 variants exhibited similar characteristics as the wild-type with highest catalytic efficiencies in the presence of lactate. Michaelis constant, Km for rPr-MnP3 mutants was far higher compared to wild-type indicating that enzymes with less affinity for Mn (II) were created by mutation of the Mn (II)-binding site. This resulted in gross decrease in catalytic efficiencies of the mutants relative to wild-type. While D186H was least efficient in tartrate, D186N showed equivalent catalytic efficiencies in both malonate (0.31 mM-1s-1) and tartrate (0.30 mM-1s-1). E40H/E44H was most effective in the presence of malonate. Our outcomes agree with the hypothesis that certain organic acids are involved in the catalytic cycle of manganese peroxidase. We conclude that mutation specific preferences for organic acid chelators have been created and histidine is a better alternative to asparagine in retaining the activity of Mn peroxidase. For practical applications, tartrate is the most for wild-type, E40H, E44H, D186H and D186N activity while malonate is the best for E40H/E44H mutant. Creation of more mutants and investigation of specific modulators of these mutant enzymes are on-going in our laboratory.

Differential Activity of the Extracellular Phenoloxidases in Different Strains of the Phytopathogenic Fungus, Microdochium nivale.

To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities (lignin peroxidase, Mn peroxidase, laccase, and tyrosinase) in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species, Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.

Electrocatalytic effect of recombinant Mn peroxidase from corn on microbiosensors to detect glucose

Our previous studies revealed that a recombinant enzyme from corn is effective and suitable for glucose monitoring. However, the challenge is to further extend our previous work by modification of a 10 μm diameter gold microelectrode, (GME), which guides this biosensor towards practical applications and in vivo analytical monitoring of glucose. The polyaniline (PANI) - gold nanoparticles (GNPs)- glucose oxidase (GOX)- plant-produced manganese peroxidase (PPMP)/GME composition showed excellent chronoamperometric (CA) response for the selective and sensitive enzymatic detection of glucose. PANI - GNPs- GOX- PPMP/GME exhibits a wide linear range of 0.001–16.0 mM, and a lower detection limit of 0.50 μM when applying CA. Linear sweep voltammetry (LSV) and cyclic voltammetry (CV) were also applied for further electrochemical characterization of the modified microelectrode to detect glucose. The biosensor selectivity was assessed by determining glucose concentrations in the presence of ascorbic acid, dopamine, aspartame, fructose, uric acid, caffeine, and 10% diet coke. As a result, the new dual enzyme microbiosensor is proposed as a tool that facilitates medical diagnosis and monitoring of glucose levels of foods.

Development of a highly sensitive glucose nanocomposite biosensor based on recombinant enzyme from corn.

Enzymes are biocatalysts that play a vital role in the production of biomolecules. Plants can be a valuable and cost-effective source for producing well-structured recombinant enzymes. Glucose is one of the most important biological molecules, providing energy to most living systems. An electrochemical method for immobilization of enzyme is promising because it is economic, generates less component waste, improves the signal-to-noise ratio, leads to a lower limit of detection, and stabilizes and protects the enzyme structure. A glucose biosensor was constructed using polyaniline (PANI) and a recombinant enzyme from corn, plant-produced manganese peroxidase (PPMP), with polymerization of aniline as a monomer in the presence of gold nanoparticles (AuNPs)-glucose oxidase (GOx), and bovine serum albumin. Using linear sweep voltammetry and cyclic voltammetry techniques, PANI-AuNPs-GOx-PPMP/Au electrode exhibited a superior sensing property with a wider linear range of 0.005-16.0mm, and a lower detection limit of 0.001 mm compared to PANI-GOx-PPMP/Au electrode and PANI-GOx-PPMP/AuNPs/Au electrode. The biosensor selectivity was assessed by determining glucose concentrations in the presence of ascorbic acid, dopamine, aspartame, and caffeine. We conclude that a plant-produced Mn peroxidase enzyme combined with conductive polymers and AuNPs results in a promising nanocomposite biosensor for detecting glucose. The use of such devices for quality control in the food industry can have a significant economic impact. © 2022 Society of Chemical Industry.

Manganese availability modifies nitrogen eutrophication signals in acidophilous temperate forests

Increases in atmospheric nitrogen (N) deposition are known to alter the ground vegetation of forest ecosystems by promoting nitrophilic plant species and displacing nitrogen-sensitive, oligotrophic species. Though there are well-known indicator species for eutrophication, there is often no direct verifiable relationship between N deposition and the strength of the eutrophication signal in the vegetation. The key reason for this sometimes lacking or weak relationship is that significant amounts of plant-available reactive N (Nr) are provided from the decay and mineralization of organic matter in the course of N cycling within the forest. The relationship is further complicated as N mineralization is also influenced by external N inputs. It has been shown repeatedly that various soil parameters related to acidity can confound a close N deposition-vegetation relationship, because acidity exerts a strong influence on the decomposition of organic matter. To further improve the understanding of the complex relationships between soil variables and vegetation composition, we analyzed vegetation and soil chemistry in acidophilous broad-leaved temperate forests of southwestern Germany, dominated by beech and oak. Our results suggest that manganese (Mn) availability likely is another factor modifying the eutrophication signal of the ground vegetation. In our analyses, the eutrophication signal in the vegetation was strongly related to high exchangeable Mn concentrations from the soil matrix and high Mn concentrations in the soil solution, in some cases to a greater extent than to parameters that reflect soil acidity. This response may be attributed to the function of Mn as a cofactor in the Mn peroxidase of white-rot fungi, which are the initiators of the decomposition of lignified organic matter. Since Mn depletion following previous acidic deposition is common in Central European forests, such a disconnection of the eutrophication signal in the vegetation from atmospheric N deposition might be widespread in forests on acidic soils.

Degradation of Synthetic Dyes with Plant-Produced Manganese Peroxidase and Commercial Laccase

Contamination of the environment directly impacts animal, plant, and human health, as well as agricultural production. A potential solution tested in this work incorporates the use of enzymes from fungi, manganese (Mn) peroxidase (MnP) and laccase, to degrade pollutants. Unfortunately, the MnP is too expensive to produce (more than US$24,000 per gram) to be used in remediation applications. MnP from a corn kernel biofactory can be produced in large amounts, and at <US$5 per gram, would be cost-effective in this application and resolve pollution issues. Two formulations of recombinant MnP from the corn kernel production system and commercial fungal laccase have been tested for degradation of synthetic dyes in an aqueous environment. Numerous concentrations of four synthetic azo or anthraquinone dyes were used. All dyes were partially or completely degraded by both enzymes.

Manganese limitations and the enhanced soil carbon sequestration of temperate rainforests

Manganese (Mn) has been identified as a regulatory bottleneck in carbon (C) turnover because of its role as an enzymatic co-factor in the oxidative decomposition of C by Mn-peroxidase (MnP). We tested this limit on decay using forest soils from coastal British Columbia with contrasting Mn concentrations. Moderately weathered soils (Brunisols) had an average 3.6-fold increase in MnP activity within the upper soil profile in comparison to highly weathered Podzols. Ordination of the Agaricomycete fungal community, which are responsible for MnP production, confirmed significant differences in assemblages between soil types for saprotrophic fungi, particularly species within Agaricales, Trechisporales and Auriculariales. Ectomycorrhizal fungi of Pseudotsuga menziesii were equally aligned with soil type and select taxa more abundant on Brunisols may have supplemented MnP activity. A laboratory incubation with an Mn amendment produced significant interactions in MnP activity by soil type. Surprisingly, MnP activity of both Brunisol substrates declined substantially with an amendment (− 56 and − 40% for forest floor and mineral soil, respectively), in contrast to Podzols (− 30 and + 26%, respectively). This inhibitory response was linked to considerable uptake of the added Mn in Brunisols, presumably by saprotrophic fungi, and underscores how Mn likely operates directly on fungi as a regulator of gene transcription for MnP production. Our study highlights a new perspective concerning the abiotic drivers underpinning the expansive soil C stocks across perhumid temperate rainforests of the Pacific Northwest.

Comparative Study of Secreted Proteins, Enzymatic Activities of Wood Degradation and Stilbene Metabolization in Grapevine Botryosphaeria Dieback Fungi.

Botryosphaeriaceae fungi are plant pathogens associated with Botryosphaeria dieback. To better understand the virulence factors of these fungi, we investigated the diversity of secreted proteins and extracellular enzyme activities involved in wood degradation and stilbene metabolization in Neofusicoccum parvum and Diplodia seriata, which are two major fungi associated with grapevine B. dieback. Regarding the analysis of proteins secreted by the two fungi, our study revealed that N. parvum, known to be more aggressive than D. seriata, was characterized by a higher quantity and diversity of secreted proteins, especially hydrolases and oxidoreductases that are likely involved in cell wall and lignin degradation. In addition, when fungi were grown with wood powder, the extracellular laccase and Mn peroxidase enzyme activities were significantly higher in D. seriata compared to N. parvum. Importantly, our work also showed that secreted Botryosphaeriaceae proteins produced after grapevine wood addition are able to rapidly metabolize the grapevine stilbenes. Overall, a higher diversity of resveratrol and piceatannol metabolization products was found with enzymes of N. parvum compared to D. seriata. This study emphasizes the diversity of secreted virulence factors found in B. dieback fungi and suggests that some resveratrol oligomers produced in grapevine wood after pathogen attack could be formed via pathogenic fungal oxidases.

A bienzymatic amperometric glucose biosensor based on using a novel recombinant Mn peroxidase from corn and glucose oxidase with a Nafion membrane

• Representation of the a) SEM image of Modified gold electrode b) Glucose biosensor membrane on the gold electrode surface (yellow), the enzyme-based layer modified with PPMP, BSA, GOx and Nafion and c) Amperometric responses of the glucose biosensor. Using a recombinant enzyme derived from corn and a simple modification, we fabricated a facile, fast, and cost-beneficial novel biosensor to measure glucose. The Nafion/Plant Produced Mn Peroxidase (PPMP)–glucose oxidase (GOx)–Bovine serum albumin (BSA)/Au electrode showed an excellent amperometric response to detect glucose. This biosensor is capable of responding to a wide range of glucose, 20.0 µM–15.0 mM, and has a lower detection limit (LOD) of 2.9 µM. The reproducibility response using six electrodes is also very substantial and indicates high capability of this biosensor to detect glucose concentrations. Selectivity of this electrode was investigated in an optimized experimental solution containing 10% diet green tea with citrus containing ascorbic acid (AA), and citric acid (CA) in a wide concentration of glucose at 0.02–14.0 mM with an LOD of 3.1 µM. We also applied linear sweep voltammetry (LSV) and this technique shows a wide range of glucose detection. The performance and strength of this novel enzyme biosensor were the simplicity, wide linear ranges, sensitivities, selectivity, and low limits of detection. We expect that the modified biosensor has the potential for monitoring various biofluids.

Mycoremediation of Soils Polluted with Trichloroethylene: First Evidence of Pleurotus Genus Effectiveness

Trichloroethylene (TCE) is a proven carcinogenic chlorinated organic compound widely used as a solvent in industrial cleaning solutions; it is easily found in the soil, air, and water and is a hazardous environmental pollutant. Most studies have attempted to remove TCE from air and water using different anaerobic bacteria species. In addition, a few have used white-rot fungi, although there are hardly any in soil. The objective of the present work is to assess TCE removal efficiency using two species of the genus Pleurotus that have not been tested before: Pleurotus ostreatus and Pleurotus eryngii, growing on a sandy loam soil. These fungi presented different intra- and extracellular enzymatic systems (chytochrome P450 (CYP450), laccase, Mn peroxidase (MnP)) capable of aerobically degrading TCE to less harmful compounds. The potential toxicity of TCE to P. ostreatus and P. eryngii was firstly tested in a TCE-spiked liquid broth (70 mg L−1 and 140 mg L−1) for 14 days. Then, both fungi were assessed for their ability to degrade the pollutant in sandy loam soil spiked with 140 mg kg−1 of TCE. P. ostreatus and P. eryngii improved the natural dissipation of TCE from soil by 44%. Extracellular enzymes were poorly expressed, but mainly in the presence of the contaminant, in accordance with the hypothesis of the involvement of CYP450.

Dispersed Variable-Retention Harvesting Mitigates N Losses on Harvested Sites in Conjunction With Changes in Soil Microbial Community Structure

As an alternative to clear-cutting, variable-retention harvesting is now standard forest management practice on the coast of British Columbia and in temperate forests globally, due to the benefits associated with maintaining mature forest species and forest structural diversity. Although there is some evidence that variable-retention harvesting, particularly single-tree (dispersed) retention will mitigate the impacts of clear-cutting on soil microbial communities and nutrient cycling, findings have been inconsistent. We examined microbial community structure (phospholipid-fatty acid), and nutrient availability (PRSTM probes) in a large (aggregated) retention patch and over three harvesting treatments: dispersed retention, clear-cut and clear-cut edge 2 years after harvest. Unlike previous studies, we did not observe elevated nitrate in the harvested areas, instead ammonium was elevated. Availability of N and other nutrients were surprisingly similar between the dispersed-retention treatment and the retention patch. The microbial community, however, was different in the clear-cut and dispersed-retention treatments, mostly due to significantly lower abundance of fungi combined with an increase in bacteria, specifically Gram-negative bacteria. This was accompanied by lower δ13CPDB value of the Gram-negative PLFA's in these treatments, suggesting the decline in mycorrhizal fungal abundance may have allowed the dominant Gram-negative bacteria to access more of the recently photosynthesized C. This shift in the microbial community composition in the dispersed-retention treatment did not appear to have a major impact on microbial functioning and nutrient availability, indicating that this harvesting practice is more effective at maintaining generic microbial functions/processes. However, as Mn levels were twice as high in the retention patch compared to the harvested treatments, indicating the other “narrow” processes (i.e., those performed by a small number of specialized microorganisms), such as lignin degradation, catalyzed by Mn peroxidase, which concomitantly removes Mn from solution, may be more sensitive to harvesting regimes. The effect of harvesting on such narrow nutrient cycling processes requires further investigation.

Ligninolytic Enzyme Production and Decolorization Capacity of Synthetic Dyes by Saprotrophic White Rot, Brown Rot, and Litter Decomposing Basidiomycetes.

An extensive screening of saprotrophic Basidiomycetes causing white rot (WR), brown rot (BR), or litter decomposition (LD) for the production of laccase and Mn-peroxidase (MnP) and decolorization of the synthetic dyes Orange G and Remazol Brilliant Blue R (RBBR) was performed. The study considered in total 150 strains belonging to 77 species. The aim of this work was to compare the decolorization and ligninolytic capacity among different ecophysiological and taxonomic groups of Basidiomycetes. WR strains decolorized both dyes most efficiently; high decolorization capacity was also found in some LD fungi. The enzyme production was recorded in all three ecophysiology groups, but to a different extent. All WR and LD fungi produced laccase, and the majority of them also produced MnP. The strains belonging to BR lacked decolorization capabilities. None of them produced MnP and the production of laccase was either very low or absent. The most efficient decolorization of both dyes and the highest laccase production was found among the members of the orders Polyporales and Agaricales. The strains with high MnP activity occurred across almost all fungal orders (Polyporales, Agaricales, Hymenochaetales, and Russulales). Synthetic dye decolorization by fungal strains was clearly related to their production of ligninolytic enzymes and both properties were determined by the interaction of their ecophysiology and taxonomy, with a more relevant role of ecophysiology. Our screening revealed 12 strains with high decolorization capacity (9 WR and 3 LD), which could be promising for further biotechnological utilization.

The effects of nutrient limitations on microbial respiration and organic matter decomposition in a Florida Spodosol as influenced by historical forest management practices

Recent investigations on the mechanistic underpinnings for nutrient regulated organic matter decomposition suggest that micronutrients may limit microbial activity in nitrogen (N) enriched forest ecosystems. However, these nutrient limitations could be complex for managed pine plantations in the US South that received macro- and micronutrient fertilization and weed control treatments. Soils and pine litter from second rotation loblolly pine stands were incubated to study the legacy effects of long-term silvicultural treatments [30+ years − Untreated Carryover (C) − Control (CC), Fertilization (CF), Fertilization + Weed control (CFW), Weed control (CW)] on microbial respiration and organic matter decomposition. These legacy treatment effects were contrasted with soils and litter corresponding to the current rotation’s actively managed treatments that were the same as in the first rotation (Actively managed retreated- C, F, FW, W). In general, both past rotation (CF: 17.9 μg C g−1 soil day−1) and current rotation fertilizer treatments (F: 22.3 μg C g−1 soil day−1) resulted in higher microbial respiration rates compared to their respective controls (CC: 11.9 μg C g−1 soil day−1; C: 12.5 μg C g−1 soil day−1), which likely reflected an inherently higher soil carbon content. Carbon normalized microbial respiration rates during the incubation period followed exponential decay patterns, with lower decay rates in fertilized soils compared to the average among treatments. Furthermore, N + phosphorus (P) additions suppressed microbial respiration in the CF and F treatments, but accelerated it in the CC and C treatments. This observation suggests that during the early stages of decomposition, N and P were limiting to microorganisms in those soils without a silvicultural treatment history. Positive microbial respiration response to added Cu for the CC and C, and added Mn for the CC, C, and W treatments suggested micronutrient limitations to microbial decomposition processes. For soils without a fertilization history, Mn peroxidase activity response to Mn addition levels followed the trend: high level > low level = No addition (CC and CW: p = 0.0032; C and W: p < 0.0001), and significantly correlated with microbial respiration rates (r = 0.63). Overall, the carryover effects from past forest management practices altered soil microbial and decomposition responses to nutrient additions, including alleviation of nutrient limitations to soil extracellular enzyme production in a Florida Spodosol.

An Italian Research Culture Collection of Wood Decay Fungi

One of the main aims of the University of Pavia mycology laboratory was to collect wood decay fungal (WDF) strains in order to deepen taxonomic studies, species distribution, officinal properties or to investigate potential applications such as biocomposite material production based on fungi. The Italian Alps, Apennines and wood plains were investigated to collect Basidiomycota basidiomata from living or dead trees. The purpose of this study was to investigate the wood decay strains of the Mediterranean area, selecting sampling sites in North and Central Italy, including forests near the Ligurian and Adriatic seas, or near the Lombardy lakes. The isolation of mycelia in pure culture was performed according to the current methodology and the identity of the strains was confirmed by molecular analyses. The strains are maintained in the Research Culture Collection MicUNIPV of Pavia University (Italy). Among the 500 WDF strains in the collection, the most interesting isolates from the Mediterranean area are: Dichomitus squalens (basidioma collected from Pinus pinea), Hericium erinaceus (medicinal mushroom), Inocutis tamaricis (white-rot agent on Tamarix trees), Perenniporia meridionalis (wood degrader through Mn peroxidase) and P. ochroleuca. In addition, strains of species related to the Mediterranean climate (e.g., Fomitiporia mediterranea and Cellulariella warnieri) were obtained from sites with a continental-temperate climate.

Degradation of Aflatoxin B1 by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of Pleurotus eryngii.

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g−1 dry matter) and low MnP activity (0.4 U g−1 dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable.

Recombinant Mn Peroxidase from Corn Grain Has an Excellent Electrocatalytic Effect in a Designed Amperometric Biosensor To Detect Hydrogen Peroxide at Low Concentrations

For the first time, we introduce a novel enzymatic biosensor using recombinant plant-produced Mn peroxidase (PPMP) extracted from corn grain to fabricate a facile, fast, and cost-beneficial biosensor for electrochemical detection of hydrogen peroxide (H2O2). The PPMP-modified gold electrode (PPMP/Au) showed excellent amperometric detection for the selective and sensitive enzymatic detection of H2O2. A wide linear range of 0.005–2.5 mM and a lower detection limit (LOD) of 1.3 μM (1.3 × 10–6 mol L–1) were observed. The newly developed biosensor also displayed high sensitivity with a detection limit of 8.2 μM using linear sweep voltammetry (LSV) and 7.0 μM when applying cyclic voltammetry. The modified electrode also showed excellent electrochemical performance for the determination of H2O2 in orange juice as low as 7.5 μM when applying LSV and 2.6 μM using an amperometric technique. It is expected that the modified gold electrode has the potential for applications in bioassays, environmental chemistry, and ...

Biodegradation of textile dyes waste water by Pleurotus eryngii

The potential of Pleurotus eryngii was studied fordecolorization ability of different types of textile dyes whichwere screened on liquid medium. During decolorization study onseven textile dyes, it was found that P. eryngii had the mostefficient decolorization on reactive Blue 21 with decolorizationpercentage (98.4%). On the other hand, the immobilized P.eryngii on wheat straw, rice straw, sugar cane waste and wheatbran had biodegradation ability of Blue 21 dye. Theimmobilization of fungus on sugar cane waste gave moreefficient decolorization of Blue 21 with decolorizationpercentage (83.5%). Also it was found that the decolorizationpercentage in case of free P. eryngii was (50.8%). The fungushas the ability to produce lignin-degrading enzymes as laccase,Mn peroxidase, peroxidase and catechol oxidase enzymes.Immobilized P. eryngii on sugar cane waste exhibited the abilityto treat industrial textile effluent, 85 to 90% reductions of variousnutrients including biological oxygen demand (BOD) andchemical oxygen demand COD were achieved and color removalreached to 96%. So, it was suggested that the immobilized P.eryngii might be applicable to textile effluent system. It wasfound that the reuse of the treated textile effluent withimmobilized P. eryngii on sugar cane waste for irrigation of Basilplant and good result similar to that obtained by tap water.

Influence of light on lignin-degrading activities of fungal genus Polyporus s. str.

Six strains belonging to five species of Polyporus (P. arcularius, P. arcularioides, P. tricholoma, P. cfr. tricholoma, and P. varius), collected from an Atlantic Forest area in Misiones (Argentina), where species usually grow exposed to high temperatures and humidity, were identified by morphological and molecular analyses. P. tricholoma (BAFC 4536) and P. arcularioides (BAFC 4534) were selected by their lignin-degrading enzyme production, their ability to produce primordial of basidiomes under submerged fermentation, and the decrease in lignin content caused in Poplar wood (up to 29% after 45 days). Among several variables evaluated with a Plackett-Burman design (glucose, copper, vanillic acid and manganese concentration, incubation period, and light incidence), the most important factor affecting laccase and Mn-peroxidase (MnP) production by both strains, was light incidence. Light induced fruit body development but diminished laccase and MnP production. Moreover, a modified isoenzymatic laccase pattern was observed, showing additional isoenzymes when fungi were cultivated under darkness and differences in optimal temperature. Although the studied strains did not produce high laccase and MnP titers (uppermost detected 4230 and 90 U L-1 , respectively), their laccases showed thermal stability and optimal temperature above 70 °C, representing an interesting source in the search of thermo-tolerant enzymes for biotechnological applications.

The degradative activity and adaptation potential of the litter-decomposing fungus Stropharia rugosoannulata.

The ability of the litter-decomposing basidiomycete Stropharia rugosoannulata DSM 11372 to degrade a wide range of structurally different environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs: phenanthrene, anthracene, fluorene, pyrene, and fluoranthene), synthetic anthraquinone dyes containing condensed aromatic rings, environmentally relevant alkylphenol and oxyethylated alkylphenol representatives, and oil was demonstrated within the present study. 9,10-Anthraquinone, phenanthrene-9,10-quinone, and 9-fluorenone were identified as products of anthracene, phenanthrene, and fluorene degradation, respectively. Fungal degradation was accompanied by the production of the ligninolytic enzymes: laccase and Mn peroxidase, suggesting their involvement in pollutant degradation. Extracellular polysaccharide(s) (EPS) and emulsifying compound(s) were concomitantly produced. EPS composed of mannose, glucose, and galactose was isolated from the cultivation medium, and its effects on catalytic properties of purified laccase from S. rugosoannulata (the dominating ligninolytic enzyme under the applied conditions) were studied. A simultaneous decrease of KM and Vmax values observed for the enzymatic oxidation of non-phenolic (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt; ABTS) and phenolic compounds (2,6-dimethoxyphenol) in presence of EPS suggest an interaction of EPS and laccase resulting in a modulation of the catalytic performance of the enzyme, which has, to the best of our knowledge, not been reported before. In line with such a modulation, the laccase-catalyzed oxidation of natural aromatic compounds (veratryl alcohol, adlerol) and environmental pollutants (the alkylphenol representative nonylphenol, the diphenylmethane derivative bisphenol A, and the PAH representative anthracene) was found to be enhanced in presence of EPS. The relevance of such effects for real environmental processes and their implications remain to be investigated.

Evaluation of 18 isolates of basidiomycetes for Lignocellulose degrading enzymes

Since fossil fuel resources are limited there is a necessity to produce alternative types of fuel that are renewable and eco-friendly. Basidiomycetes are potential sources of enzymes that can be used for biofuel production. The current study aimed to isolate basidiomycetes from Sri Lanka, screen them for lignocellulose degrading enzymes, namely cellulase, xylanase, laccase, Mn peroxidase and lignin peroxidase and study the effect of potential inducers of laccase production. Among the eighteen basidiomycetes isolated, Pycnoporus sp. produced the highest cellulase activity (0.23 FPU/ml) whereas Phlebiopsis sp. produced the highest xylanase activity (5.4 U/ml). Earliella scabrosa produced the highest laccase (91.2 U/l) and Mn peroxidase (17.5 U/l) activities. Lignin peroxidase activity was not detected from the isolates. Effect of alkali lignin, Cu 2+ and rice bran, three potential inducers, on laccase production by E. scabrosa , Pycnoporus sp. and Trametes hirsuta (M40) was studied. Results indicated that alkali lignin (2 g/l) significantly increased laccase production from Pycnoporus sp. and T. hirsuta (M40) while Cu 2+ increased laccase production from E. scabrosa and T. hirsuta at 200 μM. Use of rice bran (10 g/l) resulted in higher laccase production from E. scabrosa and Pycnoporus sp. High laccase activity (79600 U/l) was obtained from E. scabrosa by using 50 g/l of rice bran and by extending the incubation period to 18 days. The study concluded that some of the basidiomycetes isolated can produce significant lignocellulose degrading enzyme activities.