Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.     

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.     

Production of phenol-oxidizing enzymes in the interaction between white-rot fungi

The role of the phenol-oxidizing enzymes, laccase and peroxidase, was examined in the fungus-to-fungus interaction in dual cultures. Among five white-rot fungi, the following predominance in competition was observed: Pleurotus ostreatus > Trametes versicolor ≧ Pycnoporus coccineus > Ganoderma applanatum > Schizophyllum commune. Both phenol-oxidizing enzyme activities were detected markedly at the confrontation region, and under the mycelia growing over other colonies more than in other areas of the dual culture. This property was most notably observed in the P. ostreatus cultures. The fungi that produce superior active phenol-oxidizing enzymes were predominant in the competition between confronting fungi, indicating that phenol-oxidizing enzymes relate to fungus-to-fungus interaction.     

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l^–1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l^–1 and 99 mg l^–1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l^–1 and 79 mg l^–1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg^–1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.     

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

Summary 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.     

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.     

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.     

Contribution of manganese peroxidase and laccase to dye decoloration by Trametes versicolor

During dye decoloration by Trametes versicolor ATCC 20869 in modified Kirk’s medium, manganese peroxidase (MnP) and laccase were produced, but not lignin peroxidase, cellobiose dehydrogenase or manganese-independent peroxidase. Purified MnP decolorized azo dyes [amaranth, reactive black 5 (RB5) and Cibacron brilliant yellow] in Mn²⁺-dependent reactions but did not decolorize an anthraquinone dye [Remazol brilliant blue R (RBBR)]. However, the purified laccase decolorized RBBR five to ten times faster than the azo dyes and the addition of a redox mediator, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), did not alter decoloration rates. Amaranth and RB5 were decolorized the most rapidly by MnP since they have a hydroxyl group in an ortho position and a sulfonate group in the meta position relative to the azo bond. During a typical batch decoloration with the fungal culture, the ratio of laccase:MnP was 10:1 to 20:1 (based on enzyme activity) and increased to greater than 30:1 after decoloration was complete. Since MnP decolorized amaranth about 30 times more rapidly than laccase per unit of enzyme activity, MnP should have contributed more to decoloration than laccase in batch cultures.     

Lignocellulose Decomposition and Production of Ligninolytic Enzymes During Interaction of White Rot Fungi with Soil Microorganisms

Abstract Four strains of white rot fungi, including two strains of Pleurotus sp., one Dichomitus squalens, and one Ganoderma applanatum, were grown on milled straw. After colonization of the straw by the fungi, sterile or nonsterile plugs of soil were added to the fungal substrates. The influence of the sterile soil and the indigenous soil microbiota on fungal growth, overall respiration, and production of ligninolytic exoenzymes was assessed. A method for extraction of laccase from soil samples was developed. Lignocellulose decomposition, and enzyme production of D. squalens were enhanced by the presence of sterile soil. The availability of inorganic compounds such as manganese may be a trigger for this stimulation. Neither growth nor the production of laccase and manganese peroxidase (MnP) of the Pleurotus strains was markedly affected by the soil microbiota. These fungi were highly competitive with the soil microbiota. It was demonstrated for the first time that the exoenzymes of such fungi are active in nonsterile soil. Enzyme activity in the aqueous phase of soil was high as in the aqueous phase of the straw substrate. D. squalens and G. applanatum did not withstand the competition with the soil microbiota, but the mycelia associated with straw were overgrown by soil microorganisms. Correspondingly, the fungi did not penetrate the soil, decomposition of lignocellulose was impeded, and the activities of laccase and MnP decreased dramatically. Received: 2 April 1996; Accepted: 7 June 1996     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

Production of laccase, manganese peroxidase and lignin peroxidase by Brazilian marine-derived fungi

Marine-derived fungi are a potential for the search of new compounds with relevant features. Among these, the ligninolytic enzymes have potential applications in a large number of fields, including the environmental and industrial sectors. This is the work aimed to evaluate the enzymatic activities of three marine-derived fungi (Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 and Mucor racemosus CBMAI 847) under different carbon sources and salinity conditions by using statistical experimental design. MnP, LiP and laccase were detected when these fungi were cultured in malt extract, however when grown on basal medium containing glucose and wheat bran LiP was not detected and yet an increase in MnP and laccase was observed. Statistical analysis through surface responses was performed and results showed high values of MnP and laccase activities under 12.5% and 23% (w/v) salinity, highlighting the potential use of these fungi for industrial applications and in bioremediation of contaminated sites having high salt concentrations. The highest values for LiP (75376.34 UI L⁻¹), MnP (4484.30 IU L⁻¹) and laccase (898.15 UI L⁻¹) were obtained with the fungus M. racemosus CBMAI 847 and it is the first report concerning ligninolytic enzymes production by a zygomycete from this genus.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

Lignin biodegradation and ligninolytic enzyme studies during biopulping of Acacia mangium wood chips by tropical white rot fungi

White rot fungi are good lignin degraders and have the potential to be used in industry. In the present work, Phellinus sp., Daedalea sp., Trametes versicolor and Pycnoporus coccineus were selected due to their relatively high ligninolytic enzyme activity, and grown on Acacia mangium wood chips under solid state fermentation. Results obtained showed that manganese peroxidase produced is far more compared to lignin peroxidase, suggesting that MnP might be the predominating enzymes causing lignin degradation in Acacia mangium wood chips. Cellulase enzyme assays showed that no significant cellulase activity was detected in the enzyme preparation of T. versicolor and Phellinus sp. This low cellulolytic activity further suggests that these two white rot strains are of more interest in lignin degradation. The results on lignin losses showed 20–30% of lignin breakdown at 60 days of biodegradation. The highest lignin loss was found in Acacia mangium biotreated with T. versicolor after 60 days and recorded 26.9%, corresponding to the percentage of their wood weight loss recorded followed by P. coccineus. In general, lignin degradation was only significant from 20 days onwards. The overall percentage of lignin weight loss was within the range of 1.02–26.90% over the biodegradation periods. Microscopic observations conducted using scanning electron microscope showed that T. versicolor, P. coccineus, Daedalea sp. and Phellinus sp. had caused lignin degradation in Acacia mangium wood chips.     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday     

Potential of ligninolytic enzymatic complex produced by white‐rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.     

Carbon and nitrogen sources influence the ligninolytic enzyme activity of <Emphasis Type="Italic">Trametes versicolor</Emphasis>

Among carbon sources studied, cellobiose and mannitol provided the highest laccase (Lac) activity (648 and 742 U1^-1, respectively) of Trametes versicolor 775 while glucose gave maximum manganese peroxidase (MnP) and peroxidase activities (44 and 114U1^-1, respectively). Citrus fruit peel as growth substrate enhanced Lac activity 7-fold when compared to the medium with cellobiose, whereas grape vine sawdust increased MnP and peroxidase activity up to 148 and 677U1^-1, respectively.