Dye-decolorizing Peroxidase Research Articles

Elucidation of fungal dye-decolourizing peroxidase (DyP) and ligninolytic enzyme activities in decolourization and mineralization of azo dyes.

The aim of the study is to investigate the efficiency of Geotrichum candidum in the decolourization and mineralization of synthetic azo dyes. It includes screening of enzymes from G. candidum and its optimization, followed by decolourization and mineralization studies. Decolourization was observed to be maximum in methyl orange (94·6%) followed by Congo red (85%), trypan blue (70·4%) and Eriochrome Black T (55·6%) in 48h, suggesting the plausible degradation of the azo dyes by G. candidum. The enzyme activity study showed that DyP-type peroxidase has highest activity of 900mUml-1 compared to that of laccase (405mUml-1 ) and lignin peroxidase (LiP) (324mUml-1 ) at optimized pH (6) and temperature (35°C). Moreover, the rate of decolourization was found to be directly proportional to the production of laccase and LiP, unlike DyP-type peroxidase. Furthermore, mineralization study demonstrated reduction in aromatic amines, showing 20% mineralization of methyl orange. Geotrichum candidum with its enzyme system is able to efficiently decolourize and mineralize the experimental azo dyes. The efficient decolourization and mineralization of azo dyes makes G. candidum a promising alternative in the treatment of textile effluent contaminated with azo dyes.

Prokaryotic Peroxidases and Their Application in Biotechnology (Review)

This review considers papers on the diversity of the structure and functions of prokaryotic peroxidases. The most significant groups of peroxidases found in bacteria, such as catalases (Kats), catalase peroxidases (CPs), di-heme cytochrome c peroxidases (DiHCcPs), dye-decolorizing peroxidases (DyPs), are described. The data on their use in biotechnology are presented. Their participation in the biodegradation of recalcitrant substrates by microorganisms and the possibility of their use in bioremediation are discussed.

Enzymatic and genetic characterization of lignin depolymerization by Streptomyces sp. S6 isolated from a tropical environment

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pretreatment process to breakdown the recalcitrant lignin structure. This research focuses on the isolation and characterization of a lignin-degrading bacterial strain from a decaying oil palm empty fruit bunch (OPEFB). The isolated strain, identified as Streptomyces sp. S6, grew in a minimal medium with Kraft lignin (KL) as the sole carbon source. Several known ligninolytic enzyme assays were performed, and lignin peroxidase (LiP), laccase (Lac), dye-decolorizing peroxidase (DyP) and aryl-alcohol oxidase (AAO) activities were detected. A 55.3% reduction in the molecular weight (Mw) of KL was observed after 7 days of incubation with Streptomyces sp. S6 based on gel-permeation chromatography (GPC). Gas chromatography-mass spectrometry (GC-MS) also successfully highlighted the production of lignin-derived aromatic compounds, such as 3-methyl-butanoic acid, guaiacol derivatives, and 4,6-dimethyl-dodecane, after treatment of KL with strain S6. Finally, draft genome analysis of Streptomyces sp. S6 also revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin depolymerization, as well as for the mineralization of the lower molecular weight compounds, confirming the lignin degradation capability of the bacterial strain.

Exploring prospective forested wetland — Actinomycetes for biodeterioration of genotoxic textile azo dyes

disposal of synthetic textile dye effluents into waterbodies is a major cause of water pollution and undesirable toxicity on aquatic organisms affecting their metabolic and hormonal cycles. Moreover, it affects human health significantly by carcinogenic and mutagenic effects. Therefore, this study targets the biotreatment of azo dyes as a green eco-friendly tool using the tropical mangrove actinomycete, Streptomyces coelicolor strainSPR7, that manifests versatile decolourization of azo dyes. The highest decolourization rate (97.5%) was achieved using Methylene Blue (MB) within 72 h under the optimal conditions. Molecular docking analysis was accomplished by Autodock software, to discern docking score, binding pocket residues and interaction of azoreductase, DyP-type peroxidase and laccase with MB. The in vitro findings were highly correlating with in silico appraisals. UV-vis spectrometry, HPLC and FTIR were validating the MB biodecolourization and degradation. Interestingly, the strain detoxified MB viz . further validated by the Vignaradiata toxicity assay. Supplementarily, azo reductase was assessed to be the key biocatalyst for decolourization, while other two enzymes were involved in further mineralization of decolourized metabolites. Henceforth, a coupled in silico cum biological dye deterioration is a booming strategy for an economical, eco-friendly and time saving bioremediation to reuse textile wastewater.

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

Functional genomic analysis of bacterial lignin degraders: diversity in mechanisms of lignin oxidation and metabolism.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central β-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the β-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for H2O2 detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of H2O2 biosensors, since they exhibit a good dynamic response range (1–200 μM H2O2), short response times (2 s) and a superior sensitivity (1.3–1.4 A⋅M−1⋅cm−2) for H2O2, as well as selectivity and long term stability. In contrast to the solution state, 6E10 (with E188K, A142V and H125Y mutations) and 25F6 (with E188K, A142V, H125Y and G129D mutations) variants display much lower activity and are inhibited by high concentrations of H2O2 upon adsorption on an electrode. In terms of sensitivity, the bioelectrodes employing wt PpDyP and 29E4 variant outperform HRP based counterparts reported in the literature by 1–4 orders of magnitude. We propose the development of wt or 29E4 PpDyP based biosensor as a valuable alternative to devices that rely on peroxidases.

Resonance Raman view of the active site architecture in bacterial DyP-type peroxidases.

Dye decolorizing peroxidases (DyPs) are novel haem-containing peroxidases, which are structurally unrelated to classical peroxidases. They lack the highly conserved distal histidine that acts as an acid-base catalyst in the catalytic reaction of classical peroxidases, which implies distinct mechanistic properties. Despite the remarkable catalytic properties and recognized potential for biotechnology applications, the knowledge of DyP's structural features in solution, which govern the reactivity and catalysis, is lagging behind. Resonance Raman (RR) spectroscopy can reveal fine details of the active site structure in hemoproteins, reporting on the oxidation and spin state and coordination of the haem cofactor. We provide an overview of the haem binding pocket architecture of the enzymes from A, B and C DyP subfamilies, in the light of those established for classical peroxidases and search for subfamily specific features among DyPs. RR demonstrates that multiple spin populations typically co-exist in DyPs, like in the case of classical peroxidases. The haem spin/coordination state is strongly pH dependent and correlates well with the respective catalytic properties of DyPs. Unlike in the case of classical peroxidases, a surprisingly high abundance of catalytically incompetent low spin population is observed in several DyPs, and tentatively related to the alternative physiological function of these enzymes. The molecular details of active sites of DyPs, elucidated by RR spectroscopy, can furthermore guide approaches for biotechnological exploitation of these promising biocatalysts.

The ant Lasius niger is a new source of bacterial enzymes with biotechnological potential for bleaching dye

Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

The Third Generation of Artificial Dye-Decolorizing Peroxidase Rationally Designed in Myoglobin

Approaches to degradation of industrial dyes are desirable, of which bioremediation is more favorable. In addition to the use of native enzymes, rational design of artificial enzymes provides an al...

Redox thermodynamics of B-class dye-decolorizing peroxidases

With >5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors. To understand these characteristics, we have investigated the redox thermodynamics of the one-electron reduction of the ferric high-spin form of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) and the variants D143A, R232A and D143A/R232A. These distal amino acids are fully conserved in all DyPs and play important roles in Compound I formation and maintenance of the heme cavity architecture and substrate access route(s). The E°′ values of the respective redox couples Fe(III)/Fe(II) varied from −350 mV (wild-type KpDyP) to −299 mV (D143A/R232A) at pH 7.0. Variable-temperature spectroelectrochemical experiments revealed that the reduction reaction of B-class DyPs is enthalpically unfavored but entropically favored with significant differences in enthalpic and entropic contributions to E°′ between the four proteins. Molecular dynamics simulations demonstrated the impact of solvent reorganization on the entropy change during reduction reaction and revealed the dynamics and restriction of substrate access channels. Obtained data are discussed with respect to the poor peroxidase activities of B-class DyPs and compared with heme peroxidases from other (super)families as well as with chlorite dismutases, which do not react with hydrogen peroxide but share a similar fold and heme cavity architecture.

Rational design of heme enzymes for biodegradation of pollutants toward a green future.

Environmental pollutants, such as industrial dyes and halophenols, are harmful to human health, which urgently demand degradation. Bioremediation has been shown to be a cost-effective and ecofriendly approach. As reviewed herein, significant progress has been made in the last decade for biodegradation of both industrial dyes and halophenols, by engineering of native dye-decolorizing peroxidases (DyPs) and dehaloperoxidases (DHPs), and by design of artificial heme enzymes in both native and de novo protein scaffolds. The catalytic efficiency of artificial DyPs and DHPs can be rationally designed comparable to or even beyond those of natural counterparts. The enzymes are on their way from laboratory to industry and will play more crucial roles in environmental protection toward a green future.

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

BackgroundDye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.ResultAfter 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in kcat/Km) and higher H2O2 tolerance (sevenfold improvement based on IC50).ConclusionTo our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.

Degradation of the phenolic β-ether lignin model dimer and dyes by dye-decolorizing peroxidase from Bacillus amyloliquefaciens.

The dye-decolorizing peroxidase from Bacillus amyloliquefaciens, BaDyP, was identified to be an efficient catalyst for the degradation of phenolic β-ether lignin model dimer guaiacylglycerol-β-guaiacyl ether (GGE) and dyes. Efeb gene encoding BaDyP from B. amyloliquefaciens MN-13 consisted of 1257 bp and the open reading frame encoded 418 amino acids. The efeb gene was expressed in Escherichia coli BL21 and a recombinant BaDyP of 50 kDa was achieved. The BaDyP exhibited activity in oxidizing GGE and decolorizing azo and triphenylmethane dyes. At pH 4.5 and 30°C the BaDyP not only completely degraded GGE by the cleavage of β-O-4 ether bond and Cα-Cβ bond, and Cα oxidation without any oxidative mediator, but also decolorized four synthetic dyes, including congo red, bromine cresol green, eriochrome black T and crystal violet. This was achieved with decolorization rates of 65.7%, 70.62%, 80.06% and 62.09%, respectively, after 72 h of incubation. BaDyP was identified as a bacteria peroxidase with great potential for the degradation of lignin and bioremediation of dye-contamination.

Expression and Characterization of a Dye-Decolorizing Peroxidase from Pseudomonas Fluorescens Pf0-1

The consumption of dyes is increasing worldwide in line with the increase of population and demand for clothes and other colored products. However, the efficiency of dyeing processes is still poor and results in large amounts of colored effluents. It is desired to develop a portfolio of enzymes which can be used for the treatment of colored wastewaters. Herein, we used genome sequence information to discover a dye-decolorizing peroxidase (DyP) from Pseudomonas fluorescens Pf-01. Two genes putatively encoding for DyPs were identified in the respective genome and cloned for expression in Escherichia coli, of which one (PfDyP B2) could be overexpressed as a soluble protein. PfDyP B2 shows some typical features known for DyPs which includes the ability to convert dyes at the expense of hydrogen peroxide. Interestingly, t-butyl hydroperoxide could be used as an alternative substrate to hydrogen peroxide. Immobilization of PfDyP B2 in calcium-alginate beads resulted in a significant increase in stability: PfDyP B2 retains 80% of its initial activity after 2 h incubation at 50 °C, while the soluble enzyme is inactivated within minutes. PfDyP B2 was also tested with aniline and ethyl diazoacetate as substrates. Based on GC-MS analyses, 30% conversion of the starting material was achieved after 65 h at 30 °C. Importantly, this is the first report of a DyP-catalyzed insertion of a carbene into an N-H bond.

Different fungal peroxidases oxidize nitrophenols at a surface catalytic tryptophan

Dye-decolorizing peroxidase (DyP) from Auricularia auricula-judae and versatile peroxidase (VP) from Pleurotus eryngii oxidize the three mononitrophenol isomers. Both enzymes have been overexpressed in Escherichia coli and in vitro activated. Despite their very different three-dimensional structures, the nitrophenol oxidation site is located at a solvent-exposed aromatic residue in both DyP (Trp377) and VP (Trp164), as revealed by liquid chromatography coupled to mass spectrometry and kinetic analyses of nitrophenol oxidation by the native enzymes and their tryptophan-less variants (the latter showing 10–60 fold lower catalytic efficiencies).

Lignin catabolic pathways reveal unique characteristics of dye-decolorizing peroxidases in Pseudomonas putida.

Lignin is one of the largest carbon reservoirs in the environment, playing an important role in the global carbon cycle. However, lignin degradation in bacteria, especially non-model organisms, has not been well characterized either enzymatically or genetically. Here, a lignin-degrading bacterial strain, Pseudomonas putida A514, was used as the research model. Genomic and proteomic analyses suggested that two B subfamily dye-decolorizing peroxidases (DypBs) were prominent in lignin depolymerization, while the classic O2 -dependent ring cleavage strategy was utilized in central pathways to catabolize lignin-derived aromatic compounds that were funnelled by peripheral pathways. These enzymes, together with a range of transporters, sequential and expression-dose dependent regulation and stress response systems coordinated for lignin metabolism. Catalytic assays indicated these DypBs show unique Mn2+ independent lignin depolymerization activity, while Mn2+ oxidation activity is absent. Furthermore, a high synergy between DypB enzymes and A514 cells was observed to promote cell growth (5 × 1012 cfus/ml) and lignin degradation (27%). This suggested DypBs are competitive lignin biocatalysts and pinpointed limited extracellular secretion capacity as the rate-limiting factor in bacterial lignin degradation. DypB production was, therefore, optimized in recombinant strains and a 14,141-fold increase in DypB activity (56,565 U/l) was achieved, providing novel insights for lignin bioconversion.

Degradation of antifungal anthraquinone compounds is a probable physiological role of DyP secreted by Bjerkandera adusta

Alizarin is an anti-fungal compound produced by the plant, Rubia tinctorum. The parasitic fungus Bjerkandera adusta Dec 1 was cultured in potato dextrose (PD) medium with or without alizarin. Alizarin was a good substrate for the dye-decolorizing peroxidase (DyP) from B. adusta Dec 1 and hampered B. adusta growth at the early stage of plate culture. During liquid shaking culture, DyP activity in cultures supplemented with 100 μM alizarin was greater than that in controls cultured without alizarin. In particular, DyP activity per dry cell mass increased approximately 3.5-, 3.1-, and 2.9-fold at 24, 30, and 36 h after inoculation, respectively, compared with control cultures. These data suggest that alizarin stimulates the expression of DyP. Interestingly, alizarin rapidly decomposed at an early stage in culture (24–42 h) in PD medium supplemented with 100 μM alizarin. Thus, alizarin appears to induce DyP expression in B. adusta Dec 1, and this DyP, in turn, rapidly degrades alizarin. Collectively, our findings suggest that the physiological role of DyP is to degrade antifungal compounds produced by plants.

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis.

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Effective melanin degradation by a synergistic laccase-peroxidase enzyme complex for skin whitening and other practical applications.

Melanin is major cause of dark skin, which is regarded as social status in eastern Asia. As a result, researchers in cosmetic industries are developing skin whitening agents. Melanin can be decolorized by many oxidative enzymes. Laccase (CueO) from Escherichia coli and dye-decolorizing peroxidase (DyP) from Bacillus subtilis were merged with the dockerin domain of endoglucanase B from Clostridium cellulovorans. Scaffoldin has great potential to exert structural benefits that enable complementary enzyme effects. The carbohydrate binding module (CBM) in scaffoldin was replaced with the melanin binding peptide (MBP) to increase melanin binding and thereby enhance melanin degradation. The modified scaffoldin exhibits a nearly 64% increase in specific binding to melanin over that of the native scaffoldin. Laccase was used to degrade melanin via the production of hydrogen peroxide, which produced synergistic activity with peroxidase. The activity of the optimized complex was approximately 6.4-fold greater than that of laccase alone. This enzyme complex can also reduce the number of melanin granules in corneocytes. Based on these results, a recombinant enzyme complex is suitable for use in melanin degradation by next generation whitening agents in the skin cosmetics industry.