Manganese-dependent Peroxidase Research Articles

Ligninolytic enzymes can participate in a multiple response system to oxidative stress in white-rot basidiomycetes: Fomes fomentarius and Tyromyces pubescens

Abstract The effect of menadione (MQ; 2-methyl-1,4-naphtoquinone) superoxide generating agent on the biological activity of two strains of white-rot fungi, Fomes fomentarius and Tyromyces pubescens, was determined. In this study 1 mM of MQ solution was added to 10-day-old idiophasic cultures. The application of MQ to F. fomentarius and T. pubescens cultures stimulated extracellular laccase (LAC) and manganese-dependent peroxidase (MnP) activities in comparison to the control values (without MQ). In the presence of MQ the concentration of oxalic acid in the medium of both fungi was dramatically decreased. MQ treatment also caused an increase of intracellular superoxide dismutase activity, formaldehyde and glutathione disulfide level in both strains. In the case of F. fomentarius, addition of MQ enhanced catalase activity. The rate of intra- and extracellular proteolysis decreased in F. fomentarius and increased in T. pubescens MQ treated cultures.

Enzyme production and chemical alterations of Eucalyptus grandis wood during biodegradation by Ceriporiopsis subvermispora in cultures supplemented with Mn 2+, corn steep liquor and glucose

Eucalyptus grandis biodegradation by Ceriporiopsis subvermispora in solid-state cultures supplemented with corn steep liquor (CSL), glucose and Mn 2+ was evaluated. Chemical composition of biotreated wood was determined to correlate the extent of wood transformation with the levels of extracellular agents produced during fungal growth. Manganese-dependent peroxidases and xylanases were detected under all culture conditions. The lowest and the highest MnP activities were observed in cultures supplemented with Mn 2+ and CSL/glucose (178 and 1317 IU kg −1 of dry wood, respectively). In all cultures, glucan and xylan losses were lower than 3% while lignin loss varied from 2.7% to 9%. Lignin loss was correlated with the cleavage of aryl-ether linkages detected with basis on derivatization followed by reductive cleavage (DFRC) analysis. The highest lignin loss and the lowest yield of DFRC monomers were observed in cultures supplemented with Mn 2+ and CSL. This culture condition presented intermediate levels of MnP production. Oxalate production was not strongly affected by addition of culture supplements suggesting that oxalate was not a limiting agent during wood degradation by C. subvermispora. Conversely, the results suggest that a compromise between the levels of MnP and Mn 2+ was necessary to promote intense degradation of lignin revealed by DFRC analysis and lignin mass balances.

Thiobarbituric acid reactive substances, Fe3+reduction and enzymatic activities in cultures ofGanoderma australegrowing onDrimys winteriwood

Ganoderma australe is a basidiomycete responsible for a natural process of selective and extensive lignin degradation. Fatty acids, thiobarbituric acid reactive substances (TBARS), Fe3+-reduction and enzymatic activities were monitored in cultures of G. australe growing on Drimys winteri wood chips. Linoleic acid was de novo synthesized, and steadily increased during 12 weeks of cultivation. Part of the unsaturated fatty acids underwent peroxidation as TBARS accumulated with biodegradation time. TBARS accumulation was proportional to the wood weight and component losses. Manganese-dependent peroxidase and lignin peroxidase were not detected in the culture extracts, whereas laccase-induced oxidation of syringaldazine peaked after 2 weeks (104+/-9 micromol oxidized min(-1) kg(-1) of dry wood), subsequently decreasing. On the other hand, nonenzymatic Fe3+-reducing activity increased as a function of cultivation time and could be involved in the initiation of lipid peroxidation.

Mn‐peroxidase production by Panus tigrinus CBS 577.79: response surface optimisation and bioreactor comparison

AbstractThis study reports the statistical optimisation through response surface methodology of the growth medium for Panus tigrinus manganese‐dependent peroxidase (MnP) production in shaken culture. Three crucial variables, including carbon source, malonic acid and Mn2+, were optimised in a nitrogen‐limited medium. Sucrose was the best carbon source for MnP production. Mn2+ ions and malonic acid significantly stimulated MnP production at an optimal concentration of 53 mg dm−3 and 8.2 mmol dm−3, respectively, resulting in 0.83 U cm−3. Further experiments were performed in lab‐scale stirred tank (STR) and bubble‐column (BCR) reactors using the previously optimised liquid medium. BCR proved to be more adequate than STR in supporting MnP production, leading to 3700 U dm−3 after 144 h with a productivity of 25.7 U dm−3 h−1. On a comparative basis with other production data in lab‐scale reactors, these results appear to be compatible with scale transfer. Copyright © 2006 Society of Chemical Industry

Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l−1 and from 55 to 390 U l−1, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.

Optimization of manganese peroxidase production by the white rot fungus Bjerkandera sp. strain BOS55

Manganese dependent peroxidase (MnP) is the most ubiquitous peroxidase produced by white rot fungi. MnP is known to be involved in lignin degradation, biobleaching and in the oxidation of hazardous organopollutants. Bjerkandera sp. strain BOS55 is a nitrogen-unregulated white rot fungus which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. Therefore, the strain is a good candidate for use in large scale production of this enzyme. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, incubation temperature and the addition of organic acids. The fungus produced the highest level of MnP (up to 900 U 1−1) when the Mn concentration was 0.2 to 1 mM, the pH value was 5.2, and the incubation temperature was 30°C. A noteworthy finding was that MnP was also produced at lower levels in the complete absence of Mn. The addition of organic acids like glycolate, malonate, glucuronate, gluconate, 2-hydroxybutyrate to the culture medium increased the peak titres of MnP up to 1250 U 1−1. FPLC profiles indicated that the organic acids stimulated the production of all MnP isoenzymes present in the extracellular fluid of the fungus.

Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju

Pleurotus sajor-caju, strain Pl-27, produces manganese-dependent peroxidase (MnP) and laccase, but not lignin peroxidase, when grown on a defined medium with glucose as sole carbon source. MnP activity was detected in fungal cultures supplemented with both high (26 mM-N) and low (2.6 mM-N) nutrient nitrogen although higher specific activity values were recorded under the latter conditions. Conversely, laccase production was not influenced by nutrient nitrogen levels under the growth conditions adopted. Both the titre and time of appearence of MnP were also affected by the concentration of Mn in the culture medium with highest enzyme levels recorded in cultures supplemented with 15 ppm Mn. Two MnP and five laccase isoforms were identified by FPLC and gel electrophoresis.

Optimum stability conditions of pH and temperature for ligninase and manganese-dependent peroxidase from Phanerochaete chrysosporium. Application to in vitro decolorization of Poly R-478 by MnP

In the present work, the two main factors affecting enzymatic stability, i.e. pH and temperature, were analysed in order to determine the optimum ones to maintain ligninase (LiP) and manganese-dependent peroxidase (MnP) activities for prolonged periods of time. The optimum pH and temperature range obtained was around 4.2 and 34 °C for the former and 4.5 and 32 °C for the latter. Under these conditions LiP and MnP showed a half-life time of about 100 and 500 h, respectively. In addition, extracellular liquid containing mainly MnP (200 U/l) was able to decolorize about 20% of the polymeric dye Poly R-478 in 15 min. The decolorization was carried out at a pH of 4.5 (6 mM sodium malonate) and a temperature of 30 °C.

Biodegradation of the diazo dye Reactive Black 5 by a wild isolate of Candida oleophila

This work looks for a better understanding of the biodegradation of xenobiotic azo dyes mediated by yeasts. During a screening program of phenolic acid assimilating capacities it was found that a non-conventional ascomycetous yeast isolate, identified as Candida oleophila, efficiently decolorizes agar plates supplemented with the commercial textile diazo dye Reactive Black 5. Aerobic batch cultures of C. oleophila could completely decolorize up to 200 mg dye l −1, an ability not yet reported for this yeast species. Moreover, this performance has been achieved in just 24 h of incubation at 26 °C in the presence of as little as 5 g glucose l −1 and without visible signs of dye adsorption to yeast cells. It was found that decolorization occurs during the exponential growth phase and neither laccase nor manganese-dependent peroxidase activities were detected in the culture medium. As far as the decolorization mechanism is concerned, our results indirectly suggest the involvement of an azoreductase-like activity in azo bonds cleavage.

Oxalic acid, Fe 3+-reduction activity and oxidative enzymes detected in culture extracts recovered from Pinus taeda wood chips biotreated by Ceriporiopsis subvermispora

Pinus taeda wood chips were treated with the biopulping fungus, Ceriporiopsis subvermispora, under solid-state fermentation for periods varying from 7 to 90 days. Low molecular mass compounds and oxidative enzymes were extracted from biotreated wood samples. Manganese-dependent peroxidase was the main oxidative enzyme on all biodegradation periods. Aqueous extracts from biotreated wood presented decreasing pH values, oxalic acid being the major organic acid secreted by the fungus. Analysis of these extracts by gas chromatography coupled with mass spectrometry (GC/MS) revealed small amounts of fatty acids, several short-chain organic acids (C3–C6) and numerous sugar derivatives. 3-methoxy-4-hydroxy benzaldehyde, 3-methoxy-4-hydroxy benzoic acid, 3,4-dihydroxy benzoic acid and tricarboxy-benzene were also found in the wood extracts. A remarkable characteristic of the wood extracts was a strong Fe 3+-reducing ability. High Fe 3+-reducing activity and high catechol concentrations were detected in the wood extracts from the undecayed control. This reducing activity and catechol concentrations decreased during the first 7 days of biodegradation. However, from the seventh day of culturing, catechol derivatives coming from lignin degradation start to accumulate in the cultures and Fe 3+-reduction activity increased again. The Fe 3+-reduction activity observed in the wood extracts indicates that Fe 2+ would be available in solution during the wood decay process. Considering that Fe 2+ and H 2O 2 (produced by this fungus based on MnP-degradation of oxalate) were present in the wood extracts, at least some extent for degradation reactions based on Fenton-chemistry, similarly to the observed in brown-rot fungi, is supposed to occur during wood decay by C. subvermispora.

Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes

Paper and pulp mills, textile and dye-making industries and alcohol distilleries release highly colored effluents that are relatively difficult to decolorize by chemical and physical treatments. White-rot basidiomycetous fungi that produce lignin-degrading enzymes are reported to be the most efficient in decolorizing such effluents. We report here decolorization of all the three effluents by a marine fungal isolate, NIOCC # 2a cultured from decaying mangrove wood. The fungus also decolorized several synthetic dyes. Laccase was the most dominant lignin-degrading enzyme produced by this fungus with very low activities of manganese-dependent peroxidase and no lignin peroxidase activity. The growth and production of laccase was best in a medium prepared with seawater having salinity in the range of 25–30 ppt. The pH optimum for the laccase activity was 3.0 and 6.0 and the temperature optimum was 60 °C. Laccase production was increased in the presence of phenolic and non-phenolic inducers. A several fold enhancement in laccase production was found during treatment of colored effluents from textile, paper and pulp mill and distillery waste. Industrial effluents and synthetic dyes added to the growing culture of this fungus were decolorized to a great extent. The culture supernatant without the fungal biomass was also effective in decolorization of these effluents to various degrees within 6 h of incubation. Extracellular polymeric substances (EPS) produced by this fungus were also useful in decolorization of these effluents. Thus, efficiency of this fungus in decolorization of various effluents with laccase that is active at pH 3.0 and 6.0 and 60 °C in the presence of seawater has great potential in bioremediation of industrial effluents. Enhanced laccase production in the presence of industrial effluents in this fungus is an added advantage during bioremediation of effluents.

The use of spectral mapping for the study of the enzyme production of the edible mushroom Pleurotus Ostreatus

The effect of the addition of extracts of agro-industrial wastes to the culture media on the production of s-glucosidase, xylanase, laccase, manganese-dependent and independent peroxidases by the edible fungus Pleurotus ostreatus was determined. The relationship between cultivation parameters and the enzyme activities was assessed by spectral mapping technique combined with non-linear mapping. It was proved that extracts enhanced markedly the activities of laccase, manganese-dependent and independent peroxidases. Multivariate mathematical-statistical methods indicated that the enzyme activities were the highest in culture containing pepper extract. It was further demonstrated that the selectivity of the enzyme production was negligible up till 14 days of fermentation and reached the maximum at the 28 th day.

Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii

The agaric basidiomycete Nematoloma frowardii has been suggested as a good alternative for production of the extracellular ligninolytic enzyme, manganese-dependent peroxidase (MnP). Some cultural and environmental factors influencing the enzymatic activity in shaken flasks and aerated fermenter cultures were evaluated to improve the yields of the process. A low nitrogen medium (1.36 mM N added as ammonium tartrate), containing 16 g/l glucose (C/N ratio = 65.3), 2 mM Mn 2+ and inoculated with immobilized polyurethane foam mycelium, made it possible to obtain a MnP yield of 2304 nkat/l in 8 days. Under these operational conditions, the enzyme productivity in the immobilized cells of N. frowardii was 1.4 times higher than that obtained with the free fungus. In the procedure with the reusable immobilized mycelium (semi-continuous culture) as many as three subsequent 10 day batches could be fermented by using the same carrier with no loss of MnP activity.

Decolorization of an azo dye, Reactive Black 5 and MnP production by yeast isolate: Debaryomyces polymorphus

The optimum conditions for decolorization of an azo dye, C.I. Reactive Black 5 (RB5) and the kinetic characteristics of manganese-dependent peroxidase (MnP) production by yeast isolate, Debaryomyces polymorphus, were investigated. D. polymorphus could completely degrade 200 mg l −1 of non-hydrolyzed and hydrolyzed C.I. Reactive Black 5 within 24 h of cultivation at an inoculum size of 1.4 g l −1 wet cells in 50 ml medium consisting of 5 g l −1 glucose and 0.5–1.0 g l −1 ammonium sulphate (pH 5–7). In addition, the MnP activities during the cultivation were evaluated in the absence and presence of 200 mg l −1 C.I. Reactive Black 5. Maximum activity of MnP (1555.6 U l −1) was detected at 24 h cultivation in the presence of the dye, and a significant reduction of the enzyme activity was observed thereafter. The presence of C.I. Reactive Black 5 in the culture was found to be indispensable to the production of MnP by D. polymorphus. A good correlation was found between the dye degradation and the enzyme production.

Ligninolytic ability and potential biotechnology applications of the South American FungusPleurotus laciniatocrenatus

The extracellular ligninolytic enzyme system of Pleurotus laciniatocrenatus, grown under different culture conditions, was characterized and the ability of this strain to degrade different components of Eucalyptus globulus wood was determined. In shaken liquid cultures grown on a C-limited medium supplemented with yeast extract (0.1%) and peptone (0.5%), the fungus produced extracellular aryl-alcohol oxidase (Aao), laccase (Lac), manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MiP) activities, their maximum levels being, respectively, about 600, 50, 1360, and 920 pkat/mL. The supplementation of 1 mmol/L vanillic acid and 150 micromol/L CuSO4 produced an increase of Lac activity levels up to 4-fold and 68.3-fold, respectively. No significant differences were found in the levels of the other ligninolytic enzyme activities when compared to the basal medium. Solid-state fermentation cultures on E. globulus wood chips revealed Lac and MiP activities. These cultures showed degradative activity on lignin and lipophilic wood extractives.

In vitro wood decay of Eucalyptus grandis by the basidiomycete fungus Phellinus flavomarginatus

Phellinus flavomarginatus (Basidiomycetes), a white-rot fungus, has been used for the first time in studies on in vitro decomposition of Eucalyptus grandis wood under different water and aeration systems. Decomposition was judged by (i) the macroscopic and ultramicroscopic appearance of degradation, (ii) wood biomass loss, and (iii) enzyme activity (laccase, lignin peroxidase and manganese-dependent peroxidase). E. grandis chips showed white zones from the first (15 days) to the last analysis (60 days). Scanning electron microscopy demonstrated the formation of calcium oxalate crystals and hyphal penetration into pits of vessel elements and through wall fibres, without disruption or separation of fibres. Little wood biomass was lost. The enzyme analyses showed only the presence of manganese-dependent peroxidase; laccase and lignin peroxidase were not detected.

Mycoremediation: Current State and Perspectives

360 International Journal of Medicinal Mushrooms Mycoremediation (also called fungal treatment or fungal-based technology) is the application of fungi in remediation of polluted soils and aqueous effl uents. Th e fungi mostly used are wood-rot Basidiomycetes capable of degrading lignin (ligninolytic fungi). Most of these fungi cause white rot of wood, and so they are often called white-rot fungi (WRF). Th e ability of WRF to degrade lignin is due to a complex of extracellular enzymes—namely, lignin peroxidase, manganese dependent peroxidase, hydrogen peroxide generating oxidases, and phenol oxidases such as laccase. Th e lignin peroxidases were fi rst discovered in the basidiomycete Phanerochaete chrysosporium Burds., and in the 1980s this fungus was the main experimental model in lignin degradation research. Due to the nonspecifi c character of radical-mediated reactions of ligninolytic enzymes, the degradation of a wide variety of xenobiotic compounds, having an aromatic structure like lignin, has become a subject of extensive research.

Biology, Food, Medicinal, and Biotechnological Applications of the Tropical Mushroom Pleurotus tuberregium (Rumph.:Fr.) Singer

Pleurotus tuberregium (Syn. Lentinus tuberregium), a tropical Basidiomycete, is the only species of Pleurotus known to produce fruit bodies from a true sclerotium. P. tuberregium, often collected in the field growing on decaying logs, can also be cultivated on many different lignocellulosic substrates. Fruit bodies can be found growing out of the sclerotia in the wild and can be induced to do the same if subjected to soaking followed by exposure to warm temperature and humid conditions. This fungus is reported to be distributed in most of equatorial Africa, Madagascar, India, Sri Lanka, South East Asia, Papua New Guinea, New Caledonia, and Northern Australia.Until recent times, the taxonomic position of P. tuberregium has been problematic. The leathery fruit bodies and dimitic hyphal system with intercalary skeletal hyphae possessed by this fungus led authorities such as E. J. H. Corner and D. N. Pegler to place P. tuberregium in the genus Panus and Lentinus, respectively. However, more recent studies have shown P. tuberregium to have nematotoxic abilities, which supports the classification of this fungus in the genus Pleurotus by R. Singer in 1986. Molecular systematics using ribosomal DNA sequences have shown P. tuberregium to be nested with the genus Pleurotus.Phylogeny studies of P. tuberregium based on ITS sequences have shown that the African isolates studied showed very little variation, indicating a homogenous gene pool of P. tuberregium in Western Africa. In contrast, there was more gene flow in the Australasia-Pacific region, which could suggest that the origin of diversification for P. tuberregium is the Australasia-Pacific region.In nature, during the early months of the rainy season, sporophores germinating from fruit bodies discharge spores. These spores, having adequate humidity and warmth in the environment, germinate and seek compatible mates to combine with and begin the dominant dikaryotic mycelia stage in their life cycle. The life cycle of P. tuberregium is different from most other Basidiomycetes in having the sclerotial stage between the mycelia stage and fruit body formation. However, we have also observed in nature and in the laboratory that the genetic background and substrate conditions can cause some isolates of P. tuberregium to go from the mycelia stage directly to sporophore formation. Fıeld observations and laboratory studies have revealed that P. tuberregium is a white rot fungus that secretes laccase- and manganese-dependent peroxidase as its main enzymes used in lignin degradation. Cultivation studies have also shown that P. tuberregium can grow on virtually any lignocellulosic substrate to produce sclerotia and sporophores.The advantage derived from this quality is the possibility of mass producing the highly-sought-after sclerotia in West Africa, where until recent times, collection from the wild was the only source.Also, the understanding of the breeding pattern, which is tetrapolar, as well as breeding and selection studies have led to the development of improved strains tor use in commercial cultivation or this fungus. Similarly,its high temperature requirements in addition to its ability to degrade many lignocel-lulosic substrates has made it a potential candidate for bio technological approaches to waste recycling and bioremediation of recalcitrant pollutants.There are many reports that document traditional uses of P. tuberregium for food and medicine in Africa. Nutritional analysis has shown both sclerotium and sporophores of P. tuberregium to be an excellent source of protein, carbohydrates, and essential minerals. Scientific studies into the medicinal properties of P. tuberregium are active areas of research today. Extracts from sclerotia of P. tuberregium have been shown to have antibacterial and antitumor activities. Protein-bound polysaccharides isolated from P. tuberregium have been shown to have high antitumor activities against Sarcoma 180. Liquid cultivation of mycelia of P. tuberregium has been reported. Mass production of mycelia in bioreactors in developed countries and sclerotia in developing countries, where adequate environmental conditions prevail, will be the source of materials from P. tuberregium needed in medicinal products preparation and bio-technological applications.

Biodegradation of synthetic dyes by Irpex lacteus under various growth conditions

Abstract Degradability of azo-, anthraquinone- and Cu-phthalocyanine dye structures was studied using 39 strains of ligninolytic fungi, of which 60% could attack azo dyes, compared with 80–90% capable of attacking the other dyes. Irpex lacteus was shown to decolorize a number of various azo-, anthraquinone-, thiazine-, triphenylmethane- and phthalocyanine dyes at a concentration of 200 mg l −1 in stationary liquid culture. Decolorization levels after two weeks were 60–100%. Selective inhibition of manganese-dependent peroxidase (MnP) and laccase by sodium azide and n-propyl gallate indicated the involvement of MnP in decolorization of anthraquinone- and azo dyes. Immobilized on pinewood cubes, I. lacteus decolorized 100% of Remazol Brilliant Blue R (150 mg l −1 ) within six days. It also efficiently decolorized textile industry effluents containing color mixtures Drimarene Blue, Drimarene Red, Remazol Green and Acid Black, achieving 100%, 80%, 45% and 35% decolorization, respectively, within 3–5 days.

Production of lignocellulolytic enzymes by Trametes gallica and detection of polysaccharide hydrolase and laccase activities in polyacrylamide gels.

White rot fungus Trametes gallica was studied for the production of lignocellulolytic enzymes: cellulase, xylanase, laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). The results demonstrated that low-nitrogen (2.2 mM N) and surface stationary cultivation favored production of extracellular MnP. MnP activity reached 118.1 UL(-1) while T. gallica was grown in a low-nitrogen culture containing phenylalanine. However, laccase levels observed in high-nitrogen (22 mM N) agitated cultures were much greater than those seen in low-nitrogen. The N source experiments seemed to reveal that NH4+ plays an important role in inducing MnP and laccase of the fungus. Results showed that T. gallica produces a series of the lignocellulolytic enzymes, and needs high N to produce all the enzymes during solid-state fermentation of wheat straw. This paper also presents a modified zymogram procedure to detect xylanase and laccase of T. gallica in polyacrylamide gel. Xylanase in crude enzyme of T. gallica was displayed by contacting protein gel strips with xylan substrate gels and by staining with iodine. By immersing the protein gel strips in o-tolidine solution, the blue-green zones representing laccase activity were visualized against a colorless background.