Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot decay fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum.

Two filamentous fungi, the white-rot fungus Trametes versicolor and the soil fungus and potential biocontrol organism Trichoderma harzianum, have been grown in pure and mixed cultures on low-N (0.4 mM) and high-N (4 mM) defined synthetic media to determine the activities of selected wood-degrading enzymes such as cellobiase, cellulase, laccase, and peroxidases. Growth characteristics and enzyme activities were examined for potential correlations. Such correlations would allow the use of simple enzyme assays for measuring biomass development and would facilitate predictions about competitiveness of species in mixed fungal cultures. Our results show that while laccase and Poly Red-478 peroxidase activities indicate survival of the decay fungus, none of the monitored extracellular enzymes can serve as a quantitative indicator for biomass accumulation. As expected, the level of available nitrogen affected the production of the enzymes monitored: in low-N media, specific cellobiase, specific cellulase, and peroxidase activities were enhanced, while laccase activities were reduced. Most importantly, laccase activities of Trametes versicolor, and to a smaller extent, cellobiase activities of both fungi, were significantly induced in mixed cultures of Trametes versicolor and Trichoderma harzianum.     

Enzymatic Biobleaching of Two Recalcitrant Paper Dyes with Horseradish and Soybean Peroxidase

A stilbene dye (Direct Yellow 11) and a methine dye, Basazol 46L, recalcitrant to common chemical bleaches, were treated with horseradish and soybean peroxidases. Both enzymes were effective at chromophore removal. When compared to laccase in combination with a mediator (ABTS), soybean peroxidase was more effective at oxidative dye removal, especially for the methine dye.     

Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia

Summary This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer ‘ITS’ regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml^-1 within 15 days of incubation.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.     

Enzyme apparatus of the genusFusarium

The ability to degrade oligo- and polysaccharides by enzymes of the glycosidase and glucanglucan hydrolyse type, and esterase, phosphatase, proteinase, peroxidase, catalase, laccase and tyrosinase activities were tested in 35 strains of 11 sections of the genusFusarium.     

Lignin-modifying enzymes of flavodon flavus, a basidiomycete isolated from a coastal marine environment

A basidiomycetous fungus Flavodon flavus (Klotzsch) Ryvarden (strain 312), isolated from decaying sea grass from a coral lagoon off the west coast of India, mineralized nearly 24% of 14C-labeled synthetic lignin to 14CO2 in 24 days. When grown in low-nitrogen medium (2.4 mM N) this fungus produced three major classes of extracellular lignin-modifying enzymes (LMEs): manganese-dependent peroxidase (MNP), lignin peroxidase (LIP), and laccase. Low MNP and laccase activities were seen in high-nitrogen medium (24 mM N), but no LIP activity was seen. In media containing lignocellulosic substrates such as pine, poplar, or sugarcane bagasse as the sole source of carbon and nitrogen, relatively high MNP and moderate levels of laccases were seen, but LIP production either was not seen or was minimal. LME production was also seen in media prepared with artificial seawater. Fast protein liquid chromatography and isoelectric focusing resolved LMEs into four isozymes each of MNP and LIP, while laccase isozymes were resolved into two groups, one group containing seven isozymes (pIs 4 to 6) and the other group containing at least three isozymes (pIs < 3). The molecular masses of the different isozymes were 43 to 99 kDa for MNP, 40 and 41.5 kDa for LIP, and 43 and 99 kDa for laccase. F. flavus showed effective degradation of various dye pollutants in media prepared with or without artificial seawater. This is the first report on the production of all three major classes of LMEs by F. flavus and points to the bioremediation potential of this organism in terrestrial as well as marine environments.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. II. The ligninolytic enzymes of the coal-humic-acid-depolymerizing fungus Nematoloma frowardii b19

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

三种白腐真菌脱色噻嗪染料亚甲基蓝

本研究通过含亚甲基蓝染料的固体培养基,从19株白腐真菌菌株中分离获得3个脱色能力较强的菌株,其在平板上的脱色圈大小分别为7.5cm、6.8cm和5.5cm。鉴定其为：云芝栓孔菌Trametes versicolor（ZT-197）,绒毛栓孔菌Trametes pubescens（ZT-230）和亚黑管孔菌Bjerkandera fumosa（ZT-307）。其中,ZT-230对染料亚甲基蓝的脱色能力最强,可以将染料浓度为50mg/L的100mL液体培养基在6d之内100%脱色,而ZT-197和ZT-307在接种第10天时的脱色率为98%和80%。同时测定了3株白腐真菌在降解染料过程中的漆酶、锰过氧化物酶和木素过氧化物酶3种酶活力的规律：ZT-197和ZT-230均可分泌Lac和MnP两种酶,ZT-307只分泌LiP。本研究说明绒毛栓孔菌ZT-197在印染废水治理方面具有较好的应用前景。     

Biodegradation of Basic Violet 3 by <Emphasis Type="Italic">Candida krusei</Emphasis> isolated from textile wastewater

Basic Violet 3 (BV) belongs to the most important group of synthetic colorants and is used extensively in textile industries. It is considered as xenobiotic compound which is recalcitrant to biodegradation. As Candida krusei could not use BV as sole carbon source, experiments were conducted to study the effect of cosubstrates on decolorization of BV in semi synthetic medium using glucose, sucrose, lactose, maltose, yeast extract, peptone, urea and ammonium sulphate. Maximum decolorization (74%) was observed in media supplemented with sucrose. Use of sugarcane bagasse extract as sole nutrient source showed 100% decolorization of BV within 24 h under optimized condition. UV–visible, FTIR spectral analysis and HPLC analysis confirmed the biodegradation of BV. Six degradation products were isolated and identified. We propose the biodegradation pathway for BV which occurs via stepwise reduction and demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated BV species which was degraded completely. The study of the enzymes responsible for decolorization showed the activities of lignin peroxidase, lacasse, tyrosinase, NADH-DCIP reductase, MG reductase and azoreductase in cells before and after decolorization. A significant increase in activities of NADH-DCIP reductase and laccase was observed in the cells after decolorization. The yeast C. krusei could show the ability to decolorize the textile dye BV using inexpensive source like sugarcane bagasse extract for decolorization.     

Enzymatic characterization of Chilean native wood-rotting fungi for potential use in the bioremediation of polluted environments with chlorophenols

This work presents a preliminary report of a series of studies on the ability of several indigenous wood-rotting fungi from Chile to produce hydrolytic and ligninolytic enzymes and the evaluation of these native microorganism to future research on potential applications in bioremediation programs. Wood-rotting Basidiomycete fungi were collected from indigenous hardwood forest in the South of Chile. Twenty-eight strains were identified and qualitative enzymatic tests for peroxidases, laccase, tyrosinase, xylanase and cellulase production were performed in solid medium. Eleven selected strains were evaluated in liquid medium to quantify their ligninolytic enzyme production and their capacity to grow in solid medium supplemented with 2,4-dichlorophenol (2,4-DCF), 2,4,6-trichlorophenol (2,4,6-TCF) and pentachlorophenol (PCP). PCP degradation and ligninolytic enzymes production were also evaluated in liquid medium. Results showed that laccase was present in 28 of the selected strains (≈73%). Peroxidase was present in 40% and cellulase in 37% of the strains. Xilanase and tyrosinase were obtained in a smaller percentage in the strains (28% and 7%, respectively). The 11 selected strains showed high concentrations of lignin peroxidase (Lip) and manganese peroxidase (MnP). Anthracophyllum discolor (Sp4), produced LiP and MnP at 90.3 and MnP 125.5 U L⁻¹ respectively, compared to the control fungus Phanerochaete chrysosporium CECT-2798 that produced 58.1 and 118.4 U L⁻¹ of LiP and MnP. Tolerance test showed that native Chilean fungi did not present high tolerance to 2,4,6-TCF and PCP but were quite tolerant to 25 and 50 mg L⁻¹ of 2,4-DCF. However, pre-acclimatization in 2,4-DCP notably improved the growth in medium with 2,4,6-TCP and PCP. PCP in liquid medium was efficiently degraded by the fungi Anthracophyllum discolor, Lenzites betulina (Ru-30) and Galerina patagónica (Sp3), and the major MnP activity was produced by A. discolor (Sp4) (67 U L⁻¹).     

Characterization of a fungal strain isolated from a polyphenol polluted site

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.     

Phenol oxidase activity in bacteria of the genus <Emphasis Type="Italic">Azospirillum</Emphasis>

Phenol oxidase activity was detected for the first time in a number of strains belonging to various Azospirillum species. Both extracellular and intracellular activities of laccase, Mn-peroxidase, lignin peroxidase, and tyrosinase were observed. Extracellular enzymes were found to have higher activity. Significant differences in phenol oxidase activities were observed between species and strains.     

Effects of various media and supplements on laccase production by some white rot fungi

White rot fungi produce three main extracellular enzymes involved in ligninolysis; laccase, lignin peroxidase and manganese peroxidase. Though all white rot fungi do not produce all three enzymes, laccase occupies an important place in ligninolysis. The present paper reports its production by some white rot fungi; Daedalea flavida, Phlebia brevispora, Phlebia radiata and Polyporus sanguineus under different nutritional conditions. Of the various basal media tested, mineral salts malt extract broth proved to be the best medium for laccase production. Sugarcane bagasse proved to be the best laccase inducer among the various supplements added to different media.     

An alternative, banana peel-based medium used to investigate the catalytic properties of peroxidase from a fungus, Inonotus sp SP2, recently isolated in southern Chile

Recycling industrial wastes is one of the major goals of bioengineering research. Agricultural wastes are often rich in natural sources of organic and inorganic compounds. The present study investigates the use of banana peel waste as a non-conventional alternative to nitrogen-enriched glucose media for a white rot fungus (WRF), Inonotus sp. SP2, recently isolated in southern Chile. WRF are known to produce biodegrading enzymes, such as peroxidases, that can have industrial and biotechnological applications. To that end, the metabolic characteristics and catalytic properties of peroxidases produced by Inonotus sp. SP2 were compared between glucose and banana peel-based growth mediums. The results establish that this strain of WRF produces high concentrations of a Mn⁺²-dependent peroxidase, with greater enzymatic activity in extracellular fluid and crude enzyme extracted from fungus grown in banana peel and glucose media, respectively. H₂O₂ has an inhibiting effect that is greater for enzymes produced in glucose media, and greater biomass can be obtained in banana-peel based media. This demonstrates that banana peel is a suitable and more cost-effective alternative to conventional glucose-based media for the production of biodegradative enzymes, such as peroxidase. Unlike other strains of WRF, the metabolic characteristics of Inonotus sp. SP2 demonstrate that it enters secondary metabolism with the production oxidative enzymes after both carbon and nitrogen sources are depleted. This suggests that with further investigation, this strain of WRF may be useful in industrial applications that require the biodegradation of nitrogen and carbon-based wastes and recalcitrant compounds.     

Role of laccases and peroxidases in saprotrophic activities in the lichen Usnea undulata

Lichens produce various oxidoreductases including heme-containing peroxidases and the copper-containing phenol oxidases tyrosinase and laccase. Our earlier findings suggested that significant oxidoreductase activity occurs mainly in lichens from the order Peltigerales. Here we show that the non-Peltigeralean lichen Usnea can display significant activities of peroxidases and laccases. Strong evidence for the involvement of peroxidases and laccases in saprotrophic activities comes from the observation that their activities are induced by “starvation” due to prolonged dark storage, and also by treatment with soluble cellulose and lignin breakdown products. We also show that, given a quinone and chelated Fe, Usnea can produce hydroxyl radicals; these radicals contribute to the break down of carbohydrates or lignin. However, hydroxyl radical production is independent of laccase and peroxidase activity. Laccases and peroxidases are involved in other aspects of lichen biology; here we show that peroxidases, but not laccases, can break down lichen substances. Reduction in the amounts of lichen substances will reduce photoprotection, which will increase the photosynthetic capacity of thalli during winter when light intensities are low.     

Activities of ligninolytic enzymes in some white-rot basidiomycete strains after recovering from cryopreservation in liquid nitrogen

Fourteen strains of white-rot basidiomycetes belonging to eight species of two genera (Inonotus and Pholiota) were tested for their ability to maintain the production of laccase, peroxidase and manganese-dependent peroxidase (enzymes involved in lignin biodegradation) after a short-time preservation in liquid nitrogen with different cryoprotectives (glycerol, dimethyl sulfoxide). No negative effect of cryopreservation or the used cryoprotective on production of the ligninolytic enzymes was found in the fungi tested.     

Decolorization and detoxication of reactive industrial dyes by immobilized fungi <Emphasis Type="Italic">Trametes pubescens</Emphasis> and <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Trametes pubescens and Pleurotus ostreatus, immobilized on polyurethane foam cubes in bioreactors, were used to decolorize three industrial and model dyes at concentrations of 200, 1000 and 2000 ppm. Five sequential cycles were run for each dye and fungus. The activity of laccase, Mn-dependent and independent peroxidases, lignin peroxidase, and aryl-alcohol oxidase were daily monitored during the cycles and the toxicity of media containing 1000 and 2000 ppm of each dye was assessed by the Lemna minor (duckweed) ecotoxicity test. Both fungi were able to efficiently decolorize all dyes even at the highest concentration, and the duckweed test showed a significant reduction (p 相似文献   

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut.

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of peroxidase produced by the fungus Panus tigrinus

Synthesis of peroxidase and laccase by the fungus Panus tigrinus was significantly stimulated by addition of the lignocellulose substrate to the culture media. Peroxidase was isolated from the culture liquid and some properties of the enzyme were investigated. P. tigrinus peroxidase belongs to a group of extracellular peroxidases similar to the plant type peroxidases.