The synergistic ability of some wood-degrading fungi to transform lignins and lignosulfonates on various media

Summary In order to investigate the ligninolytic activity with mixed cultures of wood-degrading fungi, and the influence of various growth conditions on this activity, 50 wood-degrading fungi were tested for ligninolysis in pure culture and in pair-wise combinations according to a simple plate test recently developed in this laboratory. It was found that a synergistic degradation of lignin and of lignosulfonate was common among fungi inoculated pair-wise on lignin or lignosulfonate media; decomposition was enhanced in the zone where the two mycelia interacted. This synergistic effect was noted with pairs of two different white-rot fungi, with pairs of one white-rot and one brown-rot fungus, and with pairs of one white-rot and one soil Deuteromycete.Lignosulfonate was more susceptible to the synergistic action of pairs of fungi than was lignin. The synergistic attack on lignosulfonate was more pronounced on a meager medium than on a carbohydrate-rich one. On the contrary, the ligninolysis with pure cultures of the fungi was more pronounced on the carbohydrate-rich medium, and lignin was decomposed more easily than was lignosulfonate.     

Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

Summary The potential of a recently isolated wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the fungus proved to be efficiently capable of both biodegradation and biosorption as the major dye removal mechanisms. The extent of biodegradation was associated with the levels of the extracellular ligninolytic enzymes such as manganese peroxidase and laccase.     

Structure of the active site of lignin peroxidase isozyme H2: native enzyme, compound III, and reduced form.

The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes called lignin peroxidases which are involved in the degradation of both lignin and a number of persistent environmental pollutants. Lignin peroxidase isozyme H2, a glycosylated protein of approximately 40 kDa, contains a single heme. X-ray absorption spectroscopy (XAS) has been used to probe the local environment of the iron in the active site of resting enzyme, reduced enzyme, and compound III. For the native and reduced forms, respectively, the average Fe-pyrrole nitrogen distances are 2.055 and 2.02 A (+/- 0.015 A); the Fe-proximal nitrogen distance is 1.93 and 1.91 A (+/- 0.02 A) while the Fe-distal ligand distance is 2.17 and 2.10 A (+/- 0.03 A). Although the results are not as well-defined, the active-site structure of compound III is largely 2.02 +/- 0.015 A for the average Fe-pyrrole nitrogen distance, 1.90 +/- 0.02 for the Fe-proximal nitrogen, and 1.74 +/- 0.03 A for the Fe-distal ligand distance. The heme iron-pyrrole nitrogen distance is more expanded in ligninase H2 than in other peroxidases. The possible significance of this is discussed in relation to other heme proteins.     

Phosphorylation of lignin peroxidases from Phanerochaete chrysosporium. Identification of mannose 6-phosphate

Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K32PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K32PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed 32P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.     

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.     

Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions.

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.     

Activities of chitinolytic enzymes during primary and secondary colonization of wood by basidiomycetous fungi

The nitrogen (N) content of wood is usually suboptimal for fungal colonization. During decomposition of wood, an increasing fraction of the N becomes incorporated into fungal mycelium. Between 5 and 50% of the N in wood-degrading mycelium may be incorporated into chitin. Chitinolytic enzymes render this N available for re-utilization. Here, the activities of chitinolytic enzymes produced by wood-rotting fungi during degradation of spruce (Picea abies) wood were quantified in situ using fluorogenic 4-methylumbelliferyl substrates. A new method was developed that enables spatial quantification of enzyme activities on solid surfaces. All of the three tested fungi produced endochitinases, chitobiosidases and N-acetylhexosaminidases during colonization of wood. N-acetylhexosaminidase activity, and in some cases also chitobiosidase and endochitinase activities, were higher during secondary overgrowth of another fungus than during primary colonization of noncolonized wood. The results suggest that wood-degrading fungi degrade their own cell walls as well as the hyphae of earlier colonizers. Recycling of cell wall material within single mycelia and between fungal individuals during succession may lead to retention of N within woody debris.     

The Effect of Fungal Morphology on Ligninolytic Enzyme Production by a Recently Isolated Wood-Degrading Fungus Trichophyton Rubrum LSK-27

The morphology and ligninolytic enzyme production of a recently isolated wood-degrading fungus Trichophyton rubrum LSK-27 was investigated. In submerged cultures, the organism appeared to be an efficient manganese peroxidase (MnP) producer. When grown in baffled and unbaffled shake flasks with three different working volume/total volume ratios (WV/TV 10, 25 and 50%), the organism displayed notable morphological differences, with variations in pellet shape and size. Cultivation in baffled flasks with 25% WV/TV resulted in higher MnP and also laccase production as well as an earlier appearance of these enzymes in culture broth. However, oxygen limitation conditions inhibited MnP and laccase production and resulted in considerable changes in the morphology of this fungus.     

诱导物和金属离子对竹子白腐菌降解影响的研究

研究了4种诱导物和5种金属离子对白腐菌降解竹子的影响。结果表明这4种诱导物对木质素的降解没有明显的促进作用,低浓度的吐温80抑制纤维素的降解,降解率仅为3．057％;5种离子对木质素降解均有促进作用,一定浓度的离子明显地抑制纤维素的降解,其中Ca^2＋对纤维素降解的抑制作用最强,降解率仅为0．620％;诱导物和离子对半纤维素降解率影响较小;吐温80和Ca^2＋能显著提高半纤维素和木质素的选择系数,其中添加Ca^2＋时半纤维素和木质素选择性系数分别为66．565和49．331,初步显示：部分诱导物和金属离子可以有效影响白腐菌对竹子的选择性降解。     

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.     

The cellobiose-oxidizing enzymes CBQ and CbO as related to lignin and cellulose degradation — a review

Abstract: In this review properties of cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CbO) are presented and their possible involvement in lignin and cellulose degradation is discussed. Although these enzymes are produced by many different fungi, their importance for wood-degrading fungi is the topic here. CBQ is a FAD enzyme, while CbO also contains a heine group of the cytochrome b type. Protease activity is reported to convert CbO to CBQ. During oxidation of cellobiose (emanating from cellulose) to cellobiono-l,5-lactone, both enzymes reduce quinones produced by laccase and peroxidase during lignin degradation to the corresponding phenols. Many phenoxy and cation radicals are also reduced. Quinone reduction is more rapid than oxygen reduction, although oxygen is slowly reduced to superoxide and/or hydrogen peroxide. Thus, a more appropriate name for CbO is cellobiose dehydrogenase. CbO also reduces Fe(III) and together with hydrogen peroxide produced by the enzyme Fenton's reagent may be formed, resulting in hydroxyl radical production. This radical can degrade both lignin and cellulose, possibly indicating that cellobiose oxidase has a central role in degradation of wood by wood-degrading fungi.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

Metabolism of non-phenolic diarylpropane lignin substructure model compound by Coriolus versicolor

Abstract A lignin substructure model, 1-(4-ethoxy-3,5-dimethoxyphenyl)-2-(4-ethoxy-3-methoxyphenyl)-propane-1,3-diol(I), was actively metabolized by a white-rot fungus Coriolus versicolor in low nitrogen stationary cultures favouring the ligninolytic activity in the fungus. Cleavage of the dimer I between C_α and C_β of the propanoid side chain was the major degradative reaction by the fungus.     

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.     

Solubilization of lignin by the ruminal anaerobic fungus Neocallimastix patriciarum.

The ability of the ruminal anaerobic phycomycete Neocallimastix patriciarum to digest model lignin compounds and lignified structures in plant material was studied in batch culture. The fungus did not degrade or transform model lignin compounds that were representative of the predominant intermonomer linkages in lignin, nor did it solubilize acid detergent lignin that had been isolated from spear grass. In a stem fraction of sorghum, 33.6% of lignin was apparently solubilized by the fungus. Solubilization of ester- and either-linked phenolics accounted for 9.2% of the lignin released. The amounts of free phenolic acids detected in culture fluid were equivalent to the apparent loss of ester-linked phenolics from the sorghum substrate. However, the fungus was unable to cleave the ether bond in hydroxycinnamic acid bridges that cross-link lignin and polysaccharide. It is suggested that the majority of the solubilized lignin fraction was a lignin carbohydrate complex containing ether-linked hydroxycinnamic acids. The lignin carbohydrate complex was probably solubilized through dissolution of xylan in the lignin-xylan matrix rather than by lignin depolymerization.     

Glyoxal oxidases: their nature and properties

H₂O₂ has been found to be required for the activity of the main microbial enzymes responsible for lignin oxidative cleavage, peroxidases. Along with other small radicals, it is implicated in the early attack of plant biomass by fungi. Among the few extracellular H₂O₂-generating enzymes known are the glyoxal oxidases (GLOX). GLOX is a copper-containing enzyme, sharing high similarity at the level of active site structure and chemistry with galactose oxidase. Genes encoding GLOX enzymes are widely distributed among wood-degrading fungi especially white-rot degraders, plant pathogenic and symbiotic fungi. GLOX has also been identified in plants. Although widely distributed, only few examples of characterized GLOX exist. The first characterized fungal GLOX was isolated from Phanerochaete chrysosporium. The GLOX from Utilago maydis has a role in filamentous growth and pathogenicity. More recently, two other glyoxal oxidases from the fungus Pycnoporus cinnabarinus were also characterized. In plants, GLOX from Vitis pseudoreticulata was found to be implicated in grapevine defence mechanisms. Fungal GLOX were found to be activated by peroxidases in vitro suggesting a synergistic and regulatory relationship between these enzymes. The substrates oxidized by GLOX are mainly aldehydes generated during lignin and carbohydrates degradation. The reactions catalysed by this enzyme such as the oxidation of toxic molecules and the production of valuable compounds (organic acids) makes GLOX a promising target for biotechnological applications. This aspect on GLOX remains new and needs to be investigated.     

Lignin degradation by microorganisms: A review

Lignin is an abundant plant-based biopolymer that has found applications in a variety of industries from construction to bioethanol production. This recalcitrant branched polymer is naturally degraded by many different species of microorganisms, including fungi and bacteria. These microbial lignin degradation mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. The classes and mechanisms of different microbial lignin degradation options available in nature form the primary focus of the present review. This review first discusses the chemical building blocks of lignin and the industrial sources and applications of this multifaceted polymer. The review further places emphasis on the degradation of lignin by natural means, discussing in detail the lignin degradation activities of various fungal and bacterial species. The lignin-degrading enzymes produced by various microbial species, specifically white-rot fungi, brown-rot fungi, and bacteria, are described. In the end, possible directions for future lignin biodegradation applications and research investigations have been provided.     

A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase

BACKGROUND: The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. RESULTS: The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 A resolution using multiple isomorphous replacement including anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. CONCLUSIONS: The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V(H) domain. The haem iron is ligated by Met65 and His163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redox-active pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.