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.     

Toward a control of lignin and manganese peroxidases hypersecretion by Phanerochaete chrysosporium in agitated vessels: Evidence of the superiority of pneumatic bioreactors on mechanically agitated bioreactors

Phanerochaete chrysosporium and cultivated both mechanically agitated and pneumatic bioreactors. In the pneumatic devices, the yields of lignin and manganese peroxidases as well as extracellular protein, were considerably increased as compared with mechanically agitated bioreactors. Lignin peroxidase and manganese peroxidase activities as high as 4500 U . L(-1) and 1812 U . L(-1) respectively, were produced in an airlift bioreactor. By using enzyme markers, the secretion pathway and the respiration were shown to be dramatically activated in pneumatic bioreactors. The general metabolism of the fungus, when cultivated in the conventional fermentors, is oriented toward the synthesis of biomass at the expense of the synthesis of peroxidases. The use of pneumatic devices for the production of extracellular peroxidases by P. chrysosporium, avoids shear effects due to turbine agitator in the conventional fermentors, and provides a good example for the production of shear-sensitive metabolites. (c) 1993 John Wiley & Sons, Inc.     

Lignin peroxidase-negative mutant of the white-rot basidiomycete Phanerochaete chrysosporium

Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin----14CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H2O2-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.     

The genome of the thermoacidophilic red microalga <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> encodes a small family of secreted class III peroxidases that might be involved in cell wall modification

We report the identification of a small family of secreted class III plant peroxidases (Prx) from the genome of the unicellular thermoacidophilic red alga Galdieria sulphuraria (Cyanidiaceae). Apart from two class I ascorbate peroxidases and one cytochrome c peroxidase, the red algal genome encodes four class III plant peroxidases, thus complementing the short list of algal cell wall peroxidases (Passardi et al. in Genomics 89:567–579, 2007). We have characterized the family gene structure, analyzed the extracellular space and cell wall fraction of G. sulphuraria for the presence of peroxidase activity and used shotgun proteomics to identify candidate extracellular peroxidases. For a detailed enzymatic characterization, we have purified a secreted peroxidase (GsPrx04) from the cell-free medium using hydrophobic interaction chromatography. The enzyme proved heat and acid-stable and exhibited an apparent molecular mass of 40 kDa. Comparative genomics between endolithically growing G. sulphuraria and a close relative, the obligatory aquatic, cell wall-less Cyanidioschyzon merolae, revealed that class III peroxidases only occur in the terrestrial microalga, thus supporting the key function of these enzymes in the process of land colonization. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence database accession numbers: GsuAPX01 (EF589723), GsuAPX02 (EF589721), GsuCcP01 (EF589722), GsPrx01 (EF589724), GsPrx02 (EF589725), GsPrx03 (EF589726), and GsPrx04 (EF589727). The nomenclature of peroxidases has been adapted to PeroxiBase ().     

Methodological approaches for studying apoplastic redox activity: 2. Regulation of peroxidase activity

This work is devoted to the study of mechanisms of substrate regulation of extracellular peroxidase (ECPOX) activity at a distant stress (wounding) signal transmission from aboveground organ (leaf) of wheat (Triticum aestivum L., cv. Kazanskaya Yubileinaya) seedlings to the roots. Along with the high dianizidine peroxidase activity, the extracellular solution manifested 3,4-dihydrooxi-L-phenylalanine peroxidase, ascorbate peroxidase, and catalase activities. Dianizidine peroxidases were represented by several isoforms and had broad substrate specificity. It was found that ECPOX was released from the roots into the growing solution and its activity in the solution increased with root growth. Excision of the apical leaf parts in seedlings induced a sharp activation of root ECPOX in the growing solution. The interaction between ECPOX substrates at oxidation in two- and three-component systems is demonstrated. The role of ECPOX in the control of ROS balance in the plant cell apoplast might be determined by competitive and complementary interactions between different peroxidase substrates. Such substrate-substrate regulation of peroxidase activities may be important for stress-induced oxidative burst in plant cells.     

Plant peroxidases. Their primary, secondary and tertiary structures, and relation to cytochrome c peroxidase

The amino acid sequences of the 51% different horseradish peroxidase HRP C and turnip peroxidase TP 7 have previously been completed by us, but the three-dimensional structures are unknown. Recently the amino acid sequence and the crystal structure of yeast cytochrome c peroxidase have appeared. The three known apoperoxidases consist of 300 +/- 8 amino acid residues. The sequences have now been aligned and show 18% and 16% identity only, between the yeast peroxidase and plant peroxidase HRP C and TP 7, respectively. We show that different structural tests all support similar protein folds in plant peroxidases and yeast peroxidase and, therefore, a common evolutionary origin. The following tests support this thesis: (a) predicted helices in the plant peroxidases follow the complex pattern observed in the crystal structure of cytochrome c peroxidase; (b) their hydropathic profiles are similar and agree with observed buried and exposed peptide chain in cytochrome c peroxidase; (c) half-cystines which are distant in the amino acid sequence of plant peroxidases become spatial neighbours when fitted into the cytochrome c peroxidase model; (d) the two-domain structure proposed from limited proteolysis of apoperoxidase HRP C is observed in the crystal structure of cytochrome c peroxidase. The similarities and differences of the plant and yeast peroxidases and the reactive side chains of a plant peroxidase active site are described. The characteristics of Ca2+-binding sequences, derived from several superfamilies, are applied to predict the Ca2+-binding sequences in plant peroxidases.     

Regulation of synthesis of nitrite reductase in pea leaves: in-vivo and in-vitro studies

The cellular location of three peroxidase isoenzymes (PRX) in mature leaf tissue of Petunia and their affinity for Concanavalin A-Sepharose were investigated. The isoenzymes PRXa, PRXb and PRXc were identified by their positions in starch-gel zymograms. The fast-moving anodic and cathodic peroxidase bands, the isoenzymes PRXa and PRXc respectively, were the most active peroxidases in extracellular extracts. The molecular forms of PRXa showed a tissue-specific distribution between midrib and remaining leaf tissue. An intermediate-moving anodic peroxidase band, the isoenzyme PRXb, was the most active peroxidase released after extraction of isolated mesophyll protoplasts. Small amounts of the peroxidase isoenzymes were present in cell-wall-bound fractions. Incubation of a crude protein fraction with Concanavalin A-Sepharose showed that the isoenzyme PRXb bound more firmly to Concanavalin A-Sepharose than the isoenzymes PRXa and PRXc, of which only one molecular form bound partly. The results are discussed with respect to a possible function of one of the peroxidase isoenzymes, and a possible role of oligosaccharide chains in determining the cellular location of plant peroxidases is suggested.Abbreviations Con A Concanavalin A - PRX peroxidase (isoenzyme)     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Comparison of two lignin-degrading fungi:Phlebia radiata andPhanerochaete chrysosporium

Summary The ligninolytic enzymes ofPhlebia radiata were produced in static conditions earlier developed forPhanerochaete chrysosporium. The production pattern of lignin peroxidases resembled that ofP. chrysosporium. The extracellular proteins ofPhlebia radiata were separated by isoelectric focusing. Four proteins with acidic isoelectric points (4.15) were detected by peroxidase staining. The peroxidases ofP. radiata reacted with antibodies produced against a peroxidase ofPhanerochaete chrysosporium and vice versa. Thus the lignin peroxidases of the two fungi have major similarities despite slight differences in their isoelectric points and molecular weights. Veratryl alcohol was produced by both fungi and degraded to veratraldehyde, two lactones and a quinone by the ligninolytic cultures.     

Comparison of extracellular peroxidase- and esterase-deficient mutants of Streptomyces viridosporus T7A.

Peroxidase-deficient mutants of the lignin-degrading bacterium Streptomyces viridosporus T7A were screened for their production of acid-precipitable polymeric lignin, extracellular peroxidases and esterases, and immunoreactivities against a polyclonal antibody produced against electrophoretically purified peroxidase isoform P3 of wild-type S. viridosporus. The mutants showed diminished abilities to solubilize lignin and produce acid-precipitable polymeric lignin. Their peroxidase activities were decreased, and their esterase production patterns were altered. Western immunoblots demonstrated that the mutants produced proteins immunologically reactive with the antibody, but with different mobilities from those of wild-type proteins. These findings confirm a direct role for peroxidases in lignin solubilization. They also indicate a possible role for esterases.     

Silymarin synthesis and degradation by peroxidases of cell suspension cultures of Silybum marianum

Treatment of Silybum marianum cell cultures with methyl jasmonate elicits the production of the antihepatotoxic drug silymarin and its release into the culture medium. In this work, we investigated the involvement of peroxidases (EC 1.11.1.7; donor hydrogen peroxidase oxido-reductase) in silymarin turnover in cell cultures as well as the influence of elicitation on the activity towards several substrates. Peroxidases from cell extracts and, to a higher degree from the spent medium, used the silymarin precursors taxifolin and coniferyl alcohol as substrates. Silymarin compounds were also degraded by suspension culture peroxidases; however, the oxidation efficiency was not modified by elicitation. S. marianum peroxidases were able to catalyse the oxidative coupling of taxifolin and coniferyl alcohol to silybinins. The synthetic activity was mainly associated with the extracellular compartment and as before, methyl jasmonate did not modify oxidative coupling activity. Changes in the isoenzyme profiles were not observed in elicited cultures.     

A physiological characterization of Mn-tolerant tobacco plants selected by in vitro culture

In previous research, an in vitro stepwise procedure permitted us to obtain Nicotiana tabacum regenerated plant lines able to grow in the presence of Mn at 2 and 5 mM (Mn-tolerant plants). These plants showed several morpho-physiological and cytological differences in comparison to the Mn-sensitive regenerated plants. In particular, the number of xylem cells and the degree of lignification appeared to be influenced differently by these Mn concentrations. In the present work these Mn-tolerant and Mn-sensitive N. tabacum plants, maintained in the presence of Mn 2 and 5 mM, have been characterized with regards to the uptake of Mn and Fe, the activity of extracellular peroxidases in the stems, and the activity of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in the leaves. The leaf response to an increasing Mn concentration in the medium, corresponded a parallel decrease of Fe content. Plants tolerant of 5 mM Mn showed almost a doubling Mn content over that of the 5 mM Mn-sensitive plants. In the stem, 2 and 5 mM Mn inhibited the extracellular free peroxidases (guaiacol peroxidases) either in the Mn-tolerant plants or in the Mn-sensitive plants. In the Mn-sensitive plants treated with 2 mM Mn the activity of the peroxidases of the ionically and covalently bound wall peroxidases was also depressed. In 5 mM Mn-tolerant plants, an enhanced activity of the covalently bound wall peroxidases was observed. The effect of Mn on the covalently bound wall syringaldazine peroxidases was identical to that observed in the guaiacol peroxidases; the activity was significantly higher in the Mn-tolerant plants grown in the presence of 5 mM Mn. In the leaf, the increase of Mn content inhibited the activity of guaiacol peroxidase, ascorbate peroxidase and superoxide dismutase in the Mn-tolerant as well as in the Mn-sensitive plants. However, the effect was greater in the Mn-sensitive plants. Only glutathione reductase did not show significant variation except for the 2 mM Mn-sensitive plants, where an increased activity was detected.     

Molecular Evolution and Diversity of Lignin Degrading Heme Peroxidases in the Agaricomycetes

The plant and microbial peroxidase superfamily encompasses three classes of related protein families. Class I includes intracellular peroxidases of prokaryotic origin, class II includes secretory fungal peroxidases, including the lignin degrading enzymes manganese peroxidase (MnP), lignin peroxidase (LiP), and versatile peroxidase (VP), and class III includes the secretory plant peroxidases. Here, we present phylogenetic analyses using maximum parsimony and Bayesian methods that address the origin and diversification of class II peroxidases. Higher-level analyses used published full-length sequences from all members of the plant and microbial peroxidase superfamily, while lower-level analyses used class II sequences only, including 43 new sequences generated from Agaricomycetes (mushroom-forming fungi and relatives). The distribution of confirmed and proposed catalytic sites for manganese and aromatic compounds in class II peroxidases, including residues supposedly involved in three different long range electron transfer pathways, was interpreted in the context of phylogenies from the lower-level analyses. The higher-level analyses suggest that class II sequences constitute a monophyletic gene family within the plant and microbial peroxidase superfamily, and that they have diversified extensively in the basidiomycetes. Peroxidases of unknown function from the ascomycete Magnaporthe grisea were found to be the closest relatives of class II sequences and were selected to root class II sequences in the lower-level analyses. LiPs evidently arose only once in the Polyporales, which harbors many white-rot taxa, whereas MnPs and VPs are more widespread and may have multiple origins. Our study includes the first reports of partial sequences for MnPs in the Hymenochaetales and Corticiales.     

Fungal biodegradation and enzymatic modification of lignin

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H₂O₂-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.     

Influence of plant secondary metabolites on in vitro oxidation of methyl ferulate with cell wall peroxidases from lupine apoplast

Ionically bound cell wall peroxidases (POXs) were liberated to intercellular washing fluids (IWFs) and isolated together with other proteins and metabolites present in the apoplast of white lupine (Lupinus albus L. var. Bac) root. After separation of proteins from low molecular weight compounds, activity of peroxidases was monitored in in vitro experiments. Oxidation of methyl ferulate with H2O2 was studied in multi-component mixtures of plant metabolites. Secondary metabolites identified in IWFs or other natural products playing important roles in different physiological processes were applied as modifiers of the dehydrodimerization process during oxidation reactions performed in vitro. These were isoflavones and their conjugates, lupanine representing quinolizidine alkaloids synthesized in lupine, or other natural products such as quercetin, ascorbic, and salicylic acid. The influence of these substances on the oxidation kinetics of methyl ferulate was monitored with liquid chromatography with ultraviolet detection (LC/UV), and identification of compounds was confirmed with the liquid chromatography/mass spectroscopy (LC/MS) system. On the basis of data collected, it was possible to reveal changes in the activities of cell wall POXs. Application of the LC system permitted us to monitor, independently, quantitative changes of two or more reaction products in the mixtures. In multi-component combinations, oxidation yields of methyl ferulate by POXs were modified depending on the actual composition of the reaction mixture. We conclude that various classes of plant secondary metabolites can modify the yield of methyl ferulate oxidation by hydrogen peroxide in the presence of POX, due to interactions with the enzyme's active site (genistein) or radical scavenging properties of metabolites present in the reaction mixture.     

The Peroxidase Gene Family in Plants: A Phylogenetic Overview

The 73 class III peroxidase genes in Arabidopsis thaliana were used for surveying the evolutionary relationships among peroxidases in the plant kingdom. In Arabidopsis, the 73 genes were clustered in robust similarity groups. Comparison to peroxidases from other angiosperms showed that the diversity observed in Arabidopsis preceded the radiation of dicots, whereas some clusters were absent from grasses. Grasses contained some unique peroxidase clusters not seen in dicot plants. We found peroxidases in other major groups of land plants but not in algae. This might indicate that the class III peroxidase gene family appeared with the colonization of land by plants. The present survey may be used as a rational basis for further investigating the functional roles of class III peroxidases.     

Actinobacteria isolated from termite guts as a source of novel oxidative enzymes

A multi-faceted screening programme was designed to search for the oxidases, laccase, peroxidase and tyrosinase. Actinobacteria were selectively isolated from the paunch and colon region of the hindguts of the higher termite, Amitermes hastatus. The isolates were subjected to solid media assays (dye decolourization, melanin production and the utilization of indulin AT as sole carbon source) and liquid media assays. Eleven of the 39 strains had the ability to decolourize the dye RBBR, an indicator for the production of peroxidases in actinobacteria. Melanin production on ISP6 and ISP7 agar plates served as a good indicator for laccase and/or tyrosinase production and the ability of the strains to grow in the presence of indulin AT as a sole carbon source served as a good indicator of lignin peroxidase and/or general peroxidase production. Enzyme-producing strains were cultivated in liquid media and extracellular enzyme activities measured. Strains with the ability to produce oxidative enzymes under the conditions tested were identified to genus level by 16S rRNA gene analysis and compared to known oxidase producers. A strong relationship was observed between the environment sampled (termite guts where lignocellulose degradation occurs) and the dominant type of oxidative enzyme activity detected (laccases and peroxidases), which suggests the possibility of future targeted screening protocols linking the physical properties of the target enzymes with specific operational conditions required, such as lignocellulosic degradation in the preparation of biofuel feedstocks.     

Pleurotus ostreatus heme peroxidases: an in silico analysis from the genome sequence to the enzyme molecular structure

An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the "classical" superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively "new" peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P.?ostreatus but still to be thoroughly explored and characterized.