Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

Extracellular ligninases production and lignin degradation by Paenibacillus polymyxa.

Bacteria represent an attractive source for the isolation and identification of potentially useful microorganisms for lignin depolymerization, a process required for the use of agricultural waste. In this work, ten autochthonous bacteria isolated from straw, cow manure, and composts were characterized for potential use in the biodelignification of the waste. A comparison of the ability to degrade lignin and the efficiency of ligninolytic enzymes was performed in bacteria grown in media with lignin as a sole carbon source (LLM, 3.5g/L lignin-alkali) and in complex media supplemented with All-Ban fiber (FLM, 1.5g/L). Bacterial isolates showed different abilities to degrade lignin, they decreased the lignin concentration from 7.6 to 18.6% in LLM and from 11.1 to 44.8% in FLM. They also presented the activity of manganese peroxidase, lignin peroxidases, and laccases with different specific activities. However, strain 26 identified as Paenibacillus polymyxa by sequencing the 16S rRNA showed the highest activity of lignin peroxidase and the ability to degrade efficiently lignocellulose. In addition, P. polymyxa showed the highest potential (desirability ≥ 0.795) related to the best combination of properties to depolymerize lignin from biomass. The results suggest that P. polymyxa has a coordinated lignin degradation system constituted of lignin peroxidase, manganese peroxidase, and laccase enzymes.

Characterization of white rot fungi from wood decayed for lignin degradation.

The present study was conducted to isolate and identify white rot fungi (WRF) from wood decayed and to determine their ability to produce lignin-modifying enzymes (LMEs), specifically laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), on solid and liquid media supplemented with synthetic dyes namely 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), azure B, and phenol red. A total of 23 isolates of WRF were isolated from decayed wood and identified as eight different species namely Phanerochaete australis, Perenniporia tephropora, Lentinus squarrosulus, Ganoderma australe, Trametes polyzona, Lentinus sajor-caju, Gymnopilus dilepis, and Fomitopsis palustris based on morphological characteristics, DNA sequences of the internal transcribed spacer (ITS) region, and phylogenetic inference. The fungal isolates can be divided into four groups based on the type of LMEs produced, namely A (Lac-LiP-MnP) with 16 isolates, B (Lac-MnP) (three isolates), C (Lac) (three isolates), and D (MnP) (one isolate). This study highlights P. australis (BJ38) as the best producer of Lac and LiP, while L. squarrosulus (IPS72) is the best producer of MnP. The present study is the first reported P. australis as an efficient lignin degrader by demonstrating the highest activity of two important LMEs.

Lignin-degrading enzymes from a pathogenic canker-rot fungus Inonotus obliquus strain IO-B2.

Inonotus obliquus is a pathogenic fungus found in living trees and has been widely used as a traditional medicine for cancer therapy. Although lignocellulose-degrading enzymes are involved in the early stages of host infection, the parasitic life cycle of this fungus has not been fully understood. In this study, we aimed to investigate the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) from I. obliquus cultivated in Kirk's medium. The fungus was subjected to genome sequencing, and genes related to wood degradation were identified. The draft genome sequence of this fungus comprised 21,203 predicted protein-coding genes, of which 134 were estimated to be related to wood degradation. Among these, 47 genes associated with lignin degradation were found to have the highest number of mnp genes. Furthermore, we cloned the cDNA encoding a putative MnP, referred to as IoMnP1, and characterized its molecular structure. The results show that IoMnP1 has catalytic properties analogous to MnP. Phylogenetic analysis also confirmed that IoMnP1 was closely related to the MnPs from Pyrrhoderma noxium, Fomitiporia mediterranea, and Sanghuangporus baumii, which belong to the same family of Hymenochaetaceae. From the above results, we suggest that IoMnP1 is a member of MnPs.

Characterization of the biosurfactant production and enzymatic potential of bacteria isolated from an oil-contaminated saline soil.

Biosurfactants are amphiphilic compounds with extensive applications in oily contaminated environments to remove hydrocarbons. Moreover, enzymes such as laccase and manganese peroxidase are responsible for the oxidation of a variety of phenolic compounds and aromatic amines. Therefore, in the present study, bacteria with the potential to produce biosurfactants and enzymes (namely, laccase, manganese peroxidase, and endoglucanase carboxymethyl cellulose (CMCase)) were isolated from petroleum oil-contaminated soil. From 15 isolated bacteria, three isolates were selected as the best producers of biosurfactants according to the related tests, such as tests for surface tension reduction. These three bacteria indicated tolerance to a salinity test and were classified as resistant and very resistant. The isolates 3, 12, 13, and 14 showed positive results for the degradation of guaiacol, phenol red, and carboxymethylcellulose, as well as the decoloration of methylene blue by the creation of a clear halo around the bacterial colony. Upon the quantitation of the laccase and manganese peroxidase activities, 22.58 U/L and 21.81 U/L, respectively, were measured by isolate 13. Furthermore, CMCase activity was recorded with 0.057436 U/ml belonging to isolate 14. Bacterial strains with appreciable laccase, peroxidase, CMCase activity, and biosurfactant production potentials were identified through 16S rDNA sequence analysis as Bacillus sp. (isolate 3), Bacillus toyonensis (isolate 12), Bacillus cereus (isolate 13), and Bacillus tropicus (isolate 14), and their nucleotide sequences were deposited in the GenBank. The potentials for the industrial applicability of the biosurfactants and enzymes abound, and production needs to be optimized by the selected bacterial strains.

Improvement of saccharide yield from wood by simultaneous enzymatic delignification and saccharification using a ligninolytic enzyme and cellulase.

White-rot fungi are thought to hold promise for development of a delignification pretreatment process for wood biorefinery that is less energy-consuming than current processes. However, the reaction must take place over weeks and consumes non-neglectable amounts of saccharides. To establish a biological process for wood biorefinery would first require establishment of an enzymatic approach to delignification. Such an approach has the potential to lower costs and reduce saccharide loss. Here, we attempted enzymatic delignification reactions using manganese peroxidases (MnP), a lignin-degrading enzyme, under several reaction conditions. The delignification rate from beech wood meal (particle size <45 µm) of up to 11.0% in 48 h was reached in a MnP reaction supplemented with multiple co-oxidants, glucose, glucose oxidase (GOD) and commercial cellulase. An additional 48-h reaction using fresh MnP/co-oxidants increased the delignification rate to 14.2%. Simultaneous enzymatic delignification and saccharification, which occurs without a need for glucose supplementation, successfully improved the glucose yield to 160% of the reaction without MnP. Development of a more accurate imitation of the mechanisms of delignification that occurs in white-rot fungi has the potential to improve the monosaccharide yield resulting from simultaneous delignification and saccharification.

Two Manganese Peroxidases and a Laccase of Trametes polyzona KU-RNW027 with Novel Properties for Dye and Pharmaceutical Product Degradation in Redox Mediator-Free System.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10-30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese Peroxidase In Vitro.

Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.

Two Manganese Peroxidases and a Laccase of Trametes polyzona KU-RNW027 with Novel Properties for Dye and Pharmaceutical Product Degradation in Redox Mediator-Free System

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

Enhanced biodegradation of sediment-bound heavily weathered crude oil with ligninolytic enzymes encapsulated in calcium-alginate beads.

The degradation of crude and weathered crude oil following the application of crude and calcium-alginate encapsulated ligninolytic enzymes was studied using in situ microcosms. Changes in the chemical composition of the oil were monitored in crude enzyme extracts, as well as a sediment matrix, for as long as 70 days. Compound-specific effects of ligninolytic enzymes applied to the sediments were observed over time through changes in concentration of total petroleum hydrocarbons (TPH), polycyclic aromatic hydrocarbons (PAHs) and fractions of saturates, aromatics, resins and asphaltenes (SARA). As the oil weathered, most TPH and PAH fractions showed a rapid decrease in concentration. As sediment oil concentrations decreased following treatment with ligninolytic enzymes, the microbial population was enriched with hydrocarbon-degrading species. This trend demonstrates that the oil fractions initially not bioavailable for microbial degradation, were subsequently released to the sediment via catalytic conversion with laccase and manganese peroxidase, and the oil continues to be biodegraded by microbial populations.

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes.

BACKGROUND: Manganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP. RESULTS: Refolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status. CONCLUSIONS: Our results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.

Chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent.

Textile industry has led to severe environmental pollution and is posing a serious threat to the ecosystems. Immobilized biocatalysts have gained importance as potential bio-remediating agent. Manganese peroxidase (MnP) was immobilized onto glutaraldehyde activated chitosan beads by crosslinking and employed for the degradation and detoxification of dyes in textile effluents. The efficiency of chitosan-immobilized MnP (CI-MnP) was evaluated on the basis of decolorization, water quality improvement and toxicity reduction. Maximum color removal of 97.31% was recorded and up to 82.40%, 78.30% and 91.7% reductions in COD, TOC, and BOD were achieved, respectively. The cytotoxicity of bio-treated effluents reduced significantly and 38.46%, 43.47% and 41.83% Allium cepa root length, root count and mitotic index were increased, respectively, whereas brine shrimp nauplii death reduced up to 63.64%. Mutagenicity (Ames test) reduced up to 73.44% and 75.43% for TA98 and TA100 strains, respectively. The CI-MnP retained 60% activity after 10 repeated decolorization batches. The CI-MnP showed excellent efficiency for the bioremediation of textile effluents and can be used for the remediation of toxic agents in wastewater. The monitoring of processed wastewater using bioassays is suggested to evaluate bio-efficiency of treatment method for safe disposal of effluents into water bodies.

Trametes meyenii possesses elevated dye degradation abilities under normal nutritional conditions compared to other white rot fungi.

Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn(3+) chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.

Modulation of Cerrena unicolor laccase and manganese peroxidase production.

Among seven carbon sources tested, glycerol and glucose favored the Cerrena unicolor laccase production (18.8-20.3 U/mL); in addition, glycerol ensured the highest manganese peroxidase (MnP) activity (2 U/mL). Substitution of glycerol with the ethanol production residue (EPR) gave the highest laccase (90.1 U/mL) activity, while the walnut pericarp provided the highest MnP activity (7.4 U/mL). Supplementation of medium with 1 mM copper and 1 mM xylidine at appropriate time caused significant additive effect on laccase expression (333.2 U/mL) in shake-flask experiments. Overproduction of laccase activity (507 U/mL) and secretion of MnP activity was obtained when C. unicolor was cultivated in stirred-tank fermenter. C. unicolor showed several distinctive and attractive technological features: it is capable to synthesize high levels of oxidases under high carbon and high nitrogen conditions and it secretes high laccase activity during trophophase.

The secretome of Trametes versicolor grown on tomato juice medium and purification of the secreted oxidoreductases including a versatile peroxidase.

The present work was carried out with the aim to analyze the secretome of Trametes versicolor BAFC 2234 grown on tomato juice medium supplemented with copper and manganese. T. versicolor BAFC 2234 was selected among diverse wood dwelling agaricomycetes from Argentina by its ability to cause a strong white rot on hardwood and in addition to show high tolerance toward phenolic compounds. A considerable number of the identified proteins were related to the degradation/modification of lignocelluloses. Hydrolases, peroxidases and phenoloxidases were the most abundant enzymes produced under the above-mentioned culture conditions. The lignin-modifying oxidoreductases laccase, manganese peroxidase (MnP) and versatile peroxidase (VP) were successfully purified - the latter for the first time from T. versicolor. The native VP protein has a molecular mass of 45kDa and an isoelectric point of pH 3.7. The study clearly shows that complex plant-based media being rich in phenolics, such as tomato juice, can stimulate the secretion of a broad set of extracellular lignocellulolytic enzymes. Using such natural products as fungal culture media may give the opportunity to investigate plant biomass decomposition as well as the biodegradation of organic pollutants in an environment close to nature.

Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 microM within 90 min.

Identification of genes for lignin peroxidases and manganese peroxidases in ectomycorrhizal fungi.

• Genes for ligninolytic enzymes, normally associated with white-rot fungi, are shown to be widespread in a broad taxonomic range of ectomycorrhizal (ECM) fungi. • ECM fungi were screened for lignin peroxidase (LiP) and manganese peroxidase (MnP) genes by PCR using primers specific for known isozymes in the white-rot fungus Phanerochaete chrysosporium, with DNA sequencing used to confirm the identity of the amplified fragments. • Genes for LiPs were detected in ECM fungi representing the orders Agaricales, Aphyllophorales, Boletales, Cantharellales, Hymenochaetales, Sclerodermatales, Stereales and Thelephorales. MnP genes were detected in only Cortinarius rotundisporus and three ECM Stereales taxa. • The presence of genes for decomposer activities supports putative evolutionary relationships between ECM and saprotrophic fungi. Expression of the lignolytic genes may facilitate ECM fungal access to nutrients associated with dead plant material in soil and potentially a supplementary carbon supply. Strict functional boundaries between ECM and decomposer fungi may be less clear-cut than previously thought.