Production,partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10⁻⁶ IU/ml) and manganese peroxidase (24.11 × 10⁻⁶ IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × ⁻⁶ IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.     

Degradation of poplar wood by Fomes sclerodermeus: production of ligninolytic enzymes in sawdust of poplar and cedar

Fomes sclerodermeus is a white-rot fungus. Its production of laccase, manganese peroxidase and lignin peroxidase on sawdust-based media was evaluated. No lignin peroxidase activity was measured in any media tested. The higher production of laccase and manganese peroxidase were found on media containing poplar sawdust. F. sclerodermeus was grown on wood blocks of poplar during six months. Dry weight losses of the blocks reached a mean value of 51%. The quantification of cellulose and lignin in the 6-months incubated blocks showed losses of up to 58 and 56% for cellulose and lignin, respectively. The decay examined under microscope revealed mycelium colonizing the lumen of vessel elements, cell wall thinning and entire degradation of the radial parenchyma.     

Production of Ligninolytic Enzymes by Phlebia Floridensis

Summary The present work reports the production of laccase, lignin peroxidase and manganese peroxidase by the little studied white-rot fungus Phlebia floridensis under a variety of nutritional and physicochemical conditions. Among the different media and supplements the highest yields of laccase, lignin peroxidase and manganese peroxidase were recorded in the presence of sugarcane bagasse, wheat straw and rice straw, respectively. Laccase and manganese peroxidase activities were best expressed at a pH of 4.5 while lignin peroxidase was optimally active at a lower pH. Laccase proved to be much more thermostable as compared to the other two enzymes.     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Manganese regulation of manganese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens.

Extracellular manganese peroxidase and laccase activities were detected in cultures of Dichomitus squalens (Polyporus anceps) under conditions favoring lignin degradation. In contrast, neither extracellular lignin peroxidase nor aryl alcohol oxidase activity was detected in cultures grown under a wide variety of conditions. The mineralization of 14C-ring-, -side chain-, and -methoxy-labeled synthetic guaiacyl lignins by D. squalens and the expression of extracellular manganese peroxidase were dependent on the presence of Mn(II), suggesting that manganese peroxidase is an important component of this organism's lignin degradation system. The expression of laccase activity was independent of manganese. In contrast to previous findings with Phanerochaete chrysosporium, lignin degradation by D. squalens proceeded in the cultures containing excess carbon and nitrogen.     

Fate of Residual Lignin during Delignification of Kraft Pulp by Trametes versicolor

The fungus Trametes versicolor can delignify and brighten kraft pulps. To better understand the mechanism of this biological bleaching and the by-products formed, I traced the transformation of pulp lignin during treatment with the fungus. Hardwood and softwood kraft pulps containing ¹⁴C-labelled residual lignin were prepared by laboratory pulping of lignin-labelled aspen and spruce wood and then incubated with T. versicolor. After initially polymerizing the lignin, the fungus depolymerized it to alkali-extractable forms and then to soluble forms. Most of the labelled carbon accumulated in the water-soluble pool. The extractable and soluble products were oligomeric; single-ring aromatic products were not detected. The mineralization of the lignin carbon to CO₂ varied between experiments, up to 22% in the most vigorous cultures. The activities of the known enzymes laccase and manganese peroxidase did not account for all of the lignin degradation that took place in the T. versicolor cultures. This fungus may produce additional enzymes that could be useful in enzyme bleaching systems.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Secretion of Ligninolytic Enzymes and Mineralization of 14C-Ring-Labelled Synthetic Lignin by Three Phlebia tremellosa Strains

Production of ligninolytic enzymes by three strains of the white rot fungus Phlebia tremellosa (syn. Merulius tremellosus) was studied in bioreactor cultivation under nitrogen-limiting conditions. The Mn(II) concentration of the growth medium strongly affected the secretion patterns of lignin peroxidase and laccase. Two major lignin peroxidase isoenzymes were expressed in all strains. In addition, laccase and glyoxal oxidase were purified and characterized in one strain of P. tremellosa. In contrast, manganese peroxidase was not found in fast protein liquid chromatography profiles of extracellular proteins under either low (2.4 μM) or elevated (24 and 120 μM) Mn(II) concentrations. However, H₂O₂- and Mn-dependent phenol red-oxidizing activity was detected in cultures supplemented with higher Mn(II) levels. Mineralization rates of ¹⁴C-ring-labelled synthetic lignin (i.e., dehydrogenation polymerizate) by all strains under a low basal Mn(II) level were similar to those obtained for Phanerochaete chrysosporium and Phlebia radiata. A high manganese concentration repressed the evolution of ¹⁴CO₂ even when a chelating agent, sodium malonate, was included in the medium.     

Degradation of alkali lignin by two ascomycetes and free radical scavenging activity of the products

Biodegradation and bioconversion of extracted alkali lignin was performed under varying concentrations of carbon and nitrogen sources, by two potential Ascomycetes ligninolytic fungus isolated from soil. Fungus, F10 was identified as Aspergillus flavus, while APF4 as Emericella nidulans based upon closed similarity with their morphology and high homology in 18S rRNA gene sequences. The alkali lignin degradation was checked in term of disappearance of lignin content and colority. Selected fungus, degraded 19–41.6% of alkali lignin (0.25%, w/v) within 21 days of incubation and reduced the colority up to 14.4–21%. The activity of ligninolytic enzymes was periodically checked. During alkali lignin degradation manganese peroxidase (13.31?U/ml), lignin peroxidase (13.73?U/ml) and laccase (0.05?U/ml) activities were observed (at highest level). The alkali lignin degradation products and functional group changes in degraded lignin were analysed through gas chromatography-mass spectroscopy (GC-MS) and solid state ¹³C-NMR spectroscopy, respectively. The functional group modifications in alkali lignin moiety, alter its biochemical property, thus fungal mediated modified alkali lignin was further tested for reactive free radical scavenging potential with respect to hydroxyl, nitric oxide and superoxide radicals. Results demonstrate that the alkali lignin undergo degradation in studied nutritional conditions (high-carbon low nitrogen) and consequently increase its free radical scavenging activity up to 1–18%.     

Degradation of endocrine disrupting chemicals by genetic transformants with two lignin degrading enzymes in <Emphasis Type="Italic">Phlebia tremellosa</Emphasis>

A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.     

Production of ligninolytic enzymes and synthetic lignin mineralization by the bird's nest fungus Cyathus stercoreus

Production of ligninolytic enzymes and degradation of ¹⁴C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for ¹⁴C dehydrogenative polymerizates (DHP; 38% ¹⁴CO₂ after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% ¹⁴CO₂ after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of ¹⁴C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes. Received: 29 January 1999 / Received revision: 5 July 1999 / Accepted: 9 July 1999     

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe²⁺, with a K_m close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Laccase activities of Penicillium chrysogenum in relation to lignin degradation

 An extracellular laccase capable of oxidizing ABTS (the diammonium salt of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was detected in ligninolytic cultures of Penicillium chrysogenum. By contrast, no lignin peroxidase, manganese-dependent peroxidase or aryl-alcohol oxidase was detected at any time during culturing. Both ABTS laccase activity and mineralization of dehydrogenative polymerizate of coniferyl alcohol were regulated by the C/N ratio in the medium and partially inhibited in the presence of thioglycolic acid, suggesting that both events are associated. In the presence of several known laccase inducers neither ABTS laccase activity nor mineralization rates were enhanced. However, a new laccase was detected in P. chrysogenum, able to oxidize 2,6-dimethoxyphenol but not involved in lignin mineralization. Studies with the known ligninolytic basidiomycete Trametes villosa suggest that lignin degradation by this fungus also involves the action of laccase. Received: 6 July 1995/Received revision: 28 October 1995/Accepted: 6 November 1995     

Solubilization and Mineralization of Lignin by White Rot Fungi

The white rot fungi Lentinula edodes, Phanerochaete chrysosporium, Pleurotus sajor-caju, Flammulina velutipes, and Schizophyllum commune were grown in liquid media containing ¹⁴C-lignin-labelled wood, and the formation of water-soluble ¹⁴C-labelled products and ¹⁴CO₂, the growth of the fungi, and the activities of extracellular lignin peroxidase, manganese peroxidase, and laccase were measured. Conditions that affect the rate of lignin degradation were imposed, and both long-term (0- to 16-day) and short-term (0- to 72-h) effects on the production of the two types of product and on the activities of the enzymes were monitored. The production of ¹⁴CO₂-labelled products from the aqueous ones was also investigated. The short-term studies showed that the different conditions had different effects on the production of the two products and on the activities of the enzymes. Nitrogen sources inhibited the production of both products by all species when differences in growth could be discounted. Medium pH and manganese affected lignin degradation by the different species differently. With P. chrysosporium, the results were consistent, with lignin peroxidase playing a role in lignin solubilization and manganese peroxidase being important in subsequent CO₂ production.     

Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor

One-electron oxidation activity, as measured by ethylene generation from 2-keto-4-thiomethylbutyric acid, phenol oxidase activity, and the generation of hydroxyl radical were examined in cultures of the lignin-degrading white-rot basidiomycete fungus, Trametes (Coriolus) versicolor. The activity levels of specific lignin-degrading enzymes and cellulases, as well as the rate of wood degradation, also were examined. The fungus secreted a low-molecular-weight substance (M(r) 1000-5000) that catalyzed a redox reaction between molecular oxygen and an electron donor, to produce the hydroxyl radical via hydrogen peroxide. During wood decay, T. versicolor also produced significant amounts of laccase and lignin peroxidase, carboxymethyl cellulase, and Avicelase. The roles of the hydroxyl radical, phenol oxidases, and cellulases in wood degradation by white-rot fungi are discussed. That the hydroxyl radical produced by the low-molecular-weight substance secreted by T. versicolor results in new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase is suggested.     

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