Search

An open reading frame from the actinobacterium Mycolicibacterium smegmatis annotated as a Prostaglandin H Synthase (PGHS) was expressed with an N-terminal (his) 6 tag and purified to homogeneity. The enzyme has a monomeric molecular weight of 68.3 kD and exists as a dimer in the presence of nonionic detergent. The enzyme uses saturated and unsaturated fatty acids as substrates and catalyzes two reactions: the addition of molecular oxygen alpha to the carboxylate group to form the 2-hydroperoxy fatty acid, followed by reduction to the 2-hydroxy fatty acid. The initial reduction reaction does not require a source of electrons, but electrons must be provided from an appropriate donor such as epinephrine for the reduction reaction to go to completion. Minor reaction products one carbon atom shorter than the original fatty acid substrate are also observed; These most likely arise from the spontaneous decarboxylation of the 2-hydroperoxy fatty acid product to form an aldehyde. This dual function dioxygenase/peroxidase is unusual among the lipid dioxygenases and may represent a bacterial precursor to mammalian PGHS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

<b>Background and Objective:</b> Peroxidase (POD) is the most widely used enzyme in the manufacture of diagnostic kits, biosensors, immunohistochemistry and different industrial sectors. In this study, the POD was extracted from some local vegetables in Thailand; water mimosa. The POD was biochemically purified and characterized from water mimosa. <b>Materials and Methods:</b> The comparison of the peroxidase enzyme activity from water mimosa using Ion exchange chromatography was analyzed statistically using the Kruskal-Wallis one-way ANOVA non-parametric test. Crude extracted from water mimosa was purified by ion exchange chromatography by two techniques (DEAE-Sepharose chromatographic step and CM-Sepharose chromatographic). <b>Results:</b> The crude enzyme from water mimosa exhibited the highest peroxidase activity at 1,7458.5 U/mL. After purification, the peroxidase enzyme in the DEAE-Sepharose column showed a 1.61-fold increase in purity at a NaCl concentration of 0.0 M in 20 mM Tris-HCl buffer, pH 7.2, with a remaining yield of 46.15%. However, after DEAE-Sepharose and CM-Sepharose columns, the purity increased by 1.64-fold at a NaCl concentration of 0.0 M in 20 mM sodium acetate, pH 5.5, but the remaining yield was only 7.45%. The molecular weight of the POD enzyme was 32.3+2 kDa (n = 5) by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The enzyme activity of POD showed approximately 3,500 U/mL at pH 6.8 and the optimum temperature was 37°C. From these studies, peroxidase activities in water mimosa demonstrated a "high total activity". <b>Conclusion:</b> These results suggested that POD from water mimosa could replace horseradish peroxidase (HRP), the most used peroxidase, which is very valuable to reduce the costs of biosensors or diagnostic kit applications.

Background: Fungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn 2+ ions are yet not known., Methods: We describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus., Results: The enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn 2+ to Mn 3+ ions, suggesting a physiological function of this DyP in lignin modification. The K M value (49 µM) indicates that Mn 2+ ions bind with high affinity to the Xgr DyP protein but their subsequent oxidation into reactive Mn 3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn 2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn 2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical Xgr DyP structure. The oxidation of Mn 2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center., Conclusions: Our findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

Little is known about the degradability of mycotoxin deoxynivalenol (DON) by the spent mushroom substrate (SMS)-derived manganese peroxidase (MnP) and lignin peroxidase (LiP) and its potential. The present study investigated the growth inhibition of Fusarium graminearum KR1 and the degradation of DON by MnP and LiP extracted from SMS. The results from the 7-day treatment period showed that mycelium inhibition of F. graminearum KR1 by MnP and LiP were 23.7% and 74.7%, respectively. Deoxynivalenol production in the mycelium of F. graminearum KR1 was undetectable after treatment with 50 U/mL of MnP or LiP for 7 days. N -acetyl-D-glucosamine (GlcNAc) content and chitinase activity both increased in the hyphae of F. graminearum KR1 after treatment with MnP and LiP for 1, 3, and 6 h, respectively. At 12 h, only the LiP-treated group had higher chitinase activity and GlcNAc content than those of the control group ( p < 0.05). However, more than 60% of DON degradabilities (0.5 mg/kg, 1 h) were observed under various pH values (2.5, 4.5, and 6.5) in both MnP (50 U/g) and LiP (50 U/g) groups, while DON degradability at 1 mg/kg was 85.5% after 50 U/g of LiP treatment for 7 h in simulated pig gastrointestinal tracts. Similarly, DON degradability at 5 mg/kg was 67.1% after LiP treatment for 4.5 h in simulated poultry gastrointestinal tracts. The present study demonstrated that SMS-extracted peroxidases, particularly LiP, could effectively degrade DON and inhibit the mycelium growth of F. graminearum KR1.

In this study, peroxidase from Ziziphus jujuba was purified using ion exchange, and gel filtration chromatography resulting in an 18.9-fold enhancement of activity with a recovery of 20%. The molecular weight of Z. jujuba peroxidase was 56 kDa, as estimated by Sephacryl S-200. The purity was evaluated by SDS, which showed a single prominent band. The optimal activity of the peroxidase was achieved at pH 7.5 and 50 °C. Z. jujuba peroxidase showed catalytic efficiency (Kcat/Km) values of 25 and 43 for guaiacol and H 2 O 2 , respectively. It was completely inactivated when incubated with β-mercaptoethanol for 15 min. Hg 2+ , Zn 2+ , Cd 2+ , and NaN3 (5 mM) were effective peroxidase inhibitors, whereas Cu 2+ and Ca 2+ enhanced the peroxidase activity. The activation energy (Ea) for substrate hydrolysis was 43.89 kJ mol -1 , while the Z value and temperature quotient (Q 10 ) were found to be 17.3 °C and 2, respectively. The half-life of the peroxidase was between 117.46 and 14.15 min. For denaturation of the peroxidase, the activation energy for irreversible inactivation Ea*(d) was 120.9 kJmol -1 . Thermodynamic experiments suggested a non-spontaneous (∆G*d > 0) and endothermic reaction phase. Other thermodynamic parameters of the irreversible inactivation of the purified enzyme, such as ∆H* and ∆S*, were also studied. Based on these results, the purified peroxidase has a potential role in some industrial applications.

The present study deals with optimizing, producing, characterizing, application and techno- economic analysis of oxidative enzymes [Laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP)] from Aspergillus iizukae EAN605 in submerged fermentation process using pumpkin peels as a production substrate. The best operating parameters for producing Lac, MnP and LiP (6.15, 2.58 and 127.99 U mg -1 respectively) were recorded with 20 g 100 mL -1 of substrate, 4.6 mL 100 mL -1 of inoculum size at pH 5.5 after 10 days. The crude enzyme exhibited high stability at pH (3-9) and temperatures (20-60 °C). K m (Michaelis-Menten) of Lac, MnP and LiP crude enzyme was 2.25, 1.79 and 0.72 mM respectively. The decolourization of Remazol Brilliant Blue R by the crude enzyme was 84.84 %. The techno-economic analysis was assessed for a production unit with an annual operating time for enzymatic production and application is 7920 h/year and 100 m 3 of the capacity. The process would produce 27,000 cm 3 of crude enzyme with a price of USD 0.107 per cm 3 compared to USD 1 per cm 3 of the current commercial enzyme. The findings indicated that pumpkin peels have potential as a production substrate for oxidative enzymes from A. iizukae EAN605 and is economically feasible., Competing Interests: Declaration of Competing Interest The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Manganese peroxidases (MnP) from the white-rot fungi Phanerochaete chrysosporium catalyse the oxidation of Mn 2+ to Mn 3+ , a strong oxidizer able to oxidize a wide variety of organic compounds. Different approaches have been used to unravel the enzymatic properties and potential applications of MnP. However, these efforts have been hampered by the limited production of native MnP by fungi. Heterologous expression of MnP has been achieved in both eukaryotic and prokaryotic expression systems, although with limited production and many disadvantages in the process. Here we described a novel molecular approach for the expression and purification of manganese peroxidase isoform 1 (MnP1) from P. chrysosporium using an E. coli -expression system. The proposed strategy involved the codon optimization and chemical synthesis of the MnP1 gene for optimised expression in the E. coli T7 shuffle host. Recombinant MnP1 (rMnP1) was expressed as a fusion protein, which was recovered from solubilised inclusion bodies. rMnP1 was purified from the fusion protein using intein-based protein purification techniques and a one-step affinity chromatography. The designated strategy allowed production of an active enzyme able to oxidize guaiacol or Mn 2+ .

Latex, a milky fluid found in several plants, is widely used for many purposes, and its proteins have been investigated by researchers. Many studies have shown that latex produced by some plant species is a natural source of biologically active compounds, and many of the hydrolytic enzymes are related to health benefits. Research on the characterization and industrial and pharmaceutical utility of latex has progressed in recent years. Latex proteins are associated with plants' defense mechanisms, against attacks by fungi. In this respect, there are several biotechnological applications of antifungal proteins. Some findings reveal that antifungal proteins inhibit fungi by interrupting the synthesis of fungal cell walls or rupturing the membrane. Moreover, both phytopathogenic and clinical fungal strains are susceptible to latex proteins. The present review describes some important features of proteins isolated from plant latex which presented in vitro antifungal activities: protein classification, function, molecular weight, isoelectric point, as well as the fungal species that are inhibited by them. We also discuss their mechanisms of action., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this study, peroxidases (PODs) from three waste by-products: broad bean pods (BBP), pea pods (PP), and artichoke stems (ARS) were purified and their optimal conditions were determined for the first time. The purification process resulted in 4.32, 7.21, and 8.9% of POD recoveries for PP, ARS, and BBP, respectively. They were purified 2.12-, 32.97-, and 10-fold with specific activities of 27.26, 266.43, and 27 U/mg of protein, respectively. Analysis of their optimal conditions showed that POD purified from BBP and PP exhibited the highest activity at 60 °C temperature and pH 6 and 8 with strong affinity with catechol substrate (Km of 0.356 and 0.189 mM; Vmax of 0.08 and 0.041 μM/min for BBP and PP, respectively). The highest activity of ARS POD was obtained under the following conditions: temperature at 50 °C, pH from 6 to 8, and guaiacol as substrate (Km 0.375 mM; Vmax 0.012 μM/min). Apart from giving the opportunity for recycling the food industry wastes, the studied waste by-products could represent an alternative source of PODs that could find several applications in the biotechnological, chemical, and food industries.

Lignin peroxidase (LiP) was obtained from Pichia methanolica through heterologous expression. LiP was extracted, purified, and immobilized on Fe 3 O 4 @SiO 2 @polydopamine (PDA) nanoparticles to acquire immobilized LiP. The optimal preparation conditions for immobilized LiP were investigated. Results showed that the immobilization efficiency of immobilized LiP reached 56.37% when the enzyme amount, PDA concentration, and immobilization time were 12 mg, 1.6 mg/mL, and 12 h, respectively. Compared with free LiP, the immobilized LiP showed good thermal stability and storage stability and improved pH tolerance. It also retained more than 30% of its initial activity after 8 cycles, demonstrating its improved reusability. The immobilized LiP demonstrated efficacy of reaction of 100%, 100%, 100%, 100%, 79%, 73%, and 65% for tetracycline, dibutyl phthalate, 5-chlorophenol, phenol, phenanthrene, fluoranthene, and benzo(a)pyrene, respectively, while the inactivated immobilized LiP only adsorbed <25% of phenanthrene and fluoranthene. The dissipation of organic pollutants was a combination of degradation and adsorption, with the former playing a more important role., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The genus Agrilus comprises diverse exotic and agriculturally important wood-boring insects that have evolved efficient digestive systems. Agrilus mali Matsumara, an invasive insect, is causing extensive mortality to endangered wild apple trees in Tianshan. In this study, we present an in-depth characterization of the gut microbiota of A. mali based on high-throughput sequencing of the 16S rRNA gene and report the presence of lignocellulose-degrading bacteria. Thirty-nine operational taxonomic units (OTUs) were characterized from the larval gut. OTUs represented 6 phyla, 10 classes, 16 orders, 20 families, and 20 genera. The majority of bacterial OTUs belonged to the order Enterobacteriales which was the most abundant taxa in the larval gut. Cultivable bacteria revealed 9 OTUs that all belonged to Gammaproteobacteria. Subsequently, we examined the breakdown of plant cell-wall compounds by bacterial isolates. Among the isolates, the highest efficiency was observed in Pantoea sp., which was able to synthesize four out of the six enzymes (cellulase, cellobiase, β-xylanase, and β-gluconase) responsible for plant-cell wall degradation. One isolate identified as Pseudomonas orientalis exhibited lignin peroxidase activity. Our study provides the first characterization of the gut microbial diversity of A. mali larvae and shows that some cultivable bacteria play a significant role in the digestive tracts of larvae by providing nutritional needs.

Sweet potato (Ipomoea batatas L.) is the sixth most important food crop in the world. The industry discarded huge amount of sweet potato stems, rich of peroxidases and phenolics. A simple procedure was developed to make peroxidases and phenolics from sweet potato old stems. Dried stem powder was loaded into columns with water and eluted sequentially with water and 50% ethanol. Peroxidases (91%) were extracted in 5.5-fold water extracts and 87% phenolics were extracted in 4.4-fold ethanol extracts. Purified peroxidases powder was yielded at 3.1 g (8.6 unit/mg) per kilogram stems by PEG6000/Na 2 SO 4 aqueous two-phase purification from the water extracts (93.2% recovery), followed by ethanol precipitation and vacuum freeze-drying. The purified peroxidase had high activity in transforming tea catechins into theaflavins. Phenolics powder containing 43% phenolics and 27% flavonoids was yielded at 76.9 g per kilogram stems after vacuum-concentrating the ethanol extracts. This method can make valuable functional products using the sweet potato waste.

The biosynthetic route to the napyradiomycin family of bacterial meroterpenoids has been fully described 32 years following their original isolation and 11 years after their gene cluster discovery. The antimicrobial and cytotoxic natural products napyradiomycins A1 and B1 are produced using three organic substrates (1,3,6,8-tetrahydroxynaphthalene, dimethylallyl pyrophosphate, and geranyl pyrophosphate), and catalysis via five enzymes: two aromatic prenyltransferases (NapT8 and T9); and three vanadium dependent haloperoxidase (VHPO) homologues (NapH1, H3, and H4). Building upon the previous characterization of NapH1, H3, and T8, we herein describe the initial (NapT9, H1) and final (NapH4) steps required for napyradiomycin construction. This remarkably streamlined biosynthesis highlights the utility of VHPO enzymology in complex natural product generation, as NapH4 efficiently performs a unique chloronium-induced terpenoid cyclization to establish two stereocenters and a new carbon-carbon bond, and dual-acting NapH1 catalyzes chlorination and etherification reactions at two distinct stages of the pathway. Moreover, we employed recombinant napyradiomycin biosynthetic enzymes to chemoenzymatically synthesize milligram quantities in one pot in 1 day. This method represents a viable enantioselective approach to produce complex halogenated metabolites, like napyradiomycin B1, that have yet to be chemically synthesized.

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The K m and V max values for guaiacol, gallic acid and o‑phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (E a ) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K + , Zn 2+ , Ba 2+ , citric acid, malic acid, benzoic acid and EDTA·Na 2 , but inhibited by Cu 2+ , Ca 2+ , glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Synthesis of nanoparticles is an enzymatic reduction process in microorganisms. In the present study, a protein, lignin peroxidase has been purified by DEAE-Cellulose anion exchange chromatography and Biogel P-150 gel filtration chromatography from the cell suspension of Acinetobacter sp. SW30 responsible for the synthesis of gold nanoparticles (AuNP) and selenium nanoparticles (SeNP). The purified fraction has a specific activity of 29.4U/mg/min with 959 fold purification. Native and SDS PAGE confirmed that purified lignin peroxidase is monomeric enzyme with 97.4KDa molecular weight. The enzyme synthesized spherical crystalline AuNP (10±2nm) and amorphous SeNP (100±10nm). It has maximum activity at pH 2 and temperature 40°C, with 1.0mMKm value, when n-propanol was used as a substrate. Activity was completely inhibited by sodium thiosulphate and zinc sulphate. This is the first report on association of lignin peroxidase in the synthesis of AuNP and SeNP from Acinetobacter sp. SW30., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K d  = 2960 ± 940 μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCP interior , suggesting that the other site, TCP exterior , remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCP interior binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.

Catalase-related allene oxide synthase (cAOS) is a hemoprotein that converts a specific fatty acid hydroperoxide to an unstable allene oxide intermediate at turnover rates in the order of 1000 per second. Fatty acid allene oxides are intermediates in the formation of cyclopentenone or hydrolytic products in marine systems, most notably the prostanoid-related clavulones. Although the key catalytic amino acid residues around the active site of cAOS are the same as in true catalases, cAOS does not react with hydrogen peroxide. cAOS occurs exclusively as the N-terminal domain of a naturally occurring fusion protein with a C-terminal lipoxygenase (LOX) domain that supplies the hydroperoxide substrate. In marine invertebrates, an 8R-LOX domain converts arachidonic acid to 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) and the cAOS domain forms an 8,9-epoxy allene oxide. The fusion protein from the sea whip octocoral Plexaura homomalla is the prototypical model with crystal structures of the individual domains. The cAOS (43kDa) expresses exceptionally well in Escherichia coli, with yields of up to 100mg/L. This article describes in detail expression and assay of the P. homomalla cAOS and two methods for the preparation of its 8R-HPETE substrate. Another article in this volume focuses on the P. homomalla 8R-LOX (Gilbert, Neau, & Newcomer, 2018)., (© 2018 Elsevier Inc. All rights reserved.)

Vanadium-dependent haloperoxidases in seaweeds, cyanobacteria, fungi, and possibly phytoplankton play an important role in the release of halogenated volatile compounds in the environment. These halocarbons have effects on atmospheric chemistry since they cause ozone depletion. In this chapter, a survey is given of the different sources of these enzymes, some of their properties, the various methods to isolate them, and the bottlenecks in purification. The assays to detect and quantify haloperoxidase activity are described as well as their kinetic properties. Several practical tips and pitfalls are given which have not yet been published explicitly. Recent developments in research on structure and function of these enzymes are reviewed. Finally, the application of vanadium-dependent haloperoxidases in the biosynthesis of brominated and other compounds is discussed., (© 2018 Elsevier Inc. All rights reserved.)

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH 2 -terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The K m and V max for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Amauroderma rugosum is a wild mushroom species widely distributed in tropics and is classified under the class of Basidiomycetes. Basidiomycetes are well-known for their abilities of producing lignocellulolytic enzymes such as lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP). Different factors such as nutrient sources, incubation period and agitation affect the production of lignocellulolytic enzymes. The A. rugosum produced LiP in the medium supplemented with potato dextrose broth (PDB), 0.5% yeast and 1.0% saw dust at 26.70±3.31 U/mL. However, the LiP activity was increased to 106.32±5.32 U/mL when supplemented with 150 μm of copper (CuSO 4 ). The aqueous two-phase system (ATPS) is a simple, rapid and low cost method for primary extraction and recovery of LiP. A total of 25 systems made from five different molecular weights of polyethylene glycol (PEG)/dipotassium hydrogen phosphate (K 2 HPO 4 ) were tested. PEG 600 produced the highest top phase purification factor (P FT ) of 1.33±0.62 with yield of 72.18±8.50%. The optimization of the ATPS parameters, such as volume ratio V R , pH and crude enzyme loading are the factors controlling the phase partition. Our results showed that significant improvement (P FT of 6.26±2.87 with yield of 87.31±3.14%) of LiP recovery can be achieved by optimized the parameters., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aggregatibacter actinomycetemcomitans is an oral pathogen for aggressive periodontitis, and encodes a triheme c-containing membrane-bound enzyme, quinol peroxidase (QPO) that catalyzes peroxidase activity using quinol in the respiratory chain. In the previous work, we have characterized recombinant QPO purified from the membrane fraction of Escherichia coli harboring a plasmid containing QPO gene. Irreversible inactivation of QPO by high concentration of H2O2 exhibited pseudo-first order kinetics. Analysis of initial-rate kinetics of QPO may suggest that enzyme catalytic mechanism is explained by a Ping Pong Bi Bi system rather than sequential systems. In addition, the redox reactions of cytochrome c in the presence of several values of [Q1H2]/[Q1] were at equilibrium, and only about 2/3 of the cytochrome c of QPO is reduced at high ratios of [Q1H2]/[Q1]. These results indicated that one of the three heme c moieties of QPO is maintained in an oxidized form even at increased ratios of [Q1H2]/[Q1], suggesting that QPO is reduced in the absence of H2O2 and only two of the three heme c moieties are reduced in the presence of high concentration of the Q1H2. Product inhibition of QPO accorded with our theoretical model for the reaction mechanism. Considered together, the enzymatic kinetics data for QPO confirm the Ping Pong Bi Bi system., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl2 and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k cat = 17 s–1; k cat/K m = 855 mM–1 s–1) has been 100–300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.

The work shows the ability of cultured Basidiomycetes of different taxonomic groups-Lentinus edodes, Pleurotus ostreatus, Ganoderma lucidum, and Grifola frondosa-to recover gold, silver, selenium, and silicon, to elemental state with nanoparticles formation. It examines the effect of these metal and metalloid compounds on the parameters of growth and accumulation of biomass; the optimal cultivation conditions and concentrations of the studied ion-containing compounds for recovery of nanoparticles have been identified. Using the techniques of transmission electron microscopy, dynamic light scattering, X-ray fluorescence and X-ray phase analysis, the degrees of oxidation of the bioreduced elements, the ζ-potential of colloidal solutions uniformity, size, shape, and location of the nanoparticles in the culture fluid, as well as on the surface and the inside of filamentous hyphae have been determined. The study has found the part played by homogeneous chromatographically pure fungal phenol-oxidizing enzymes (laccases, tyrosinases, and Mn-peroxidases) in the recovery mechanism with formation of electrostatically stabilized colloidal solutions. A hypothetical mechanism of gold(III) reduction from HAuCl 4 to gold(0) by phenol oxidases with gold nanoparticles formation of different shapes and sizes has been introduced.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Manganese peroxidase, MnP-Tra-48424, was purified and characterized from the white-rot fungus Trametes sp.48424. MnP-Tra-48424 was strongly resistant to metal ions such as Ni 2+ , Li + , Ca 2+ , K + , Mn 2+ . MnP-Tra-48424 was also resistant to organic solvents such as propanediol, glycerol, and glycol. MnP-Tra-48424 decolorized dyes (indigo, anthraquinone, azo and triphenylmethane) and degraded different polycyclic aromatic hydrocarbons (PAHs). Indigo Carmine, Remazol Brilliant Blue R, Remazol Brilliant Violet 5R and Methyl Green were efficiently decolorized by MnP-Tra-48424. MnP-Tra-48424 also decolorized Indigo Carmine and Methyl Green combined with metal ions and organic solvents. The decolorization capability of MnP-Tra-48424 was not inhibited by selected metal ions and organic solvents. A combination of MnP-Tra-48424 and Lac-Tra-48424 improved the decolorization rate. In addition to dyes, MnP-Tra-48424 was effective at degrading individual PAHs (fluorene, fluoranthene, pyrene, phenanthrene, anthracene) and also PAHs in mixtures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: Melanin is a black or brown phenolic polymer present mainly in skin and hair. Although melanin can be degraded by some microbial species, the melanin degradation capacity of Geotrichum sp. is unknown. The aim of this study was to characterize a melanin biodegradation enzyme from Geotrichum sp., Methods: In this study, we assessed the melanin degradation activity of Geotrichum sp. in comparison with the major melanin-degrading enzymes, manganese-dependent peroxidase (MnP), manganese-independent peroxidase, lignin peroxidase and laccase. Furthermore, the effect of several carbohydrates on melanin degradation by Geotrichum sp. was determined. The MnP enzyme was purified using ammonium sulphate precipitation and Sephadex G-200 column chromatography, and then the conditions for optimal enzymatic activity were determined by adjusting the pH, temperature and Tween-80 concentration., Results: Compared with extracellular ligninolytic enzymes of Geotrichum sp., MnP had the highest ligninolytic enzyme activity; and the highest enzymatic activity was observed in the presence of glucose. The final purified MnP enzyme exhibited 6 U mL -1 activity and had a molecular weight of 54.2 kDa. The enzymatic activity was highest at pH 4.5 and 25-35°C in the absence of Tween-80., Conclusion: These results indicate the potential of MnP purified from Geotrichum sp. as a skin-lightening agent in the cosmetic industry., (© 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The use of oxidoredutive enzymes in removing organic pollutants has been the subject of much research. The oxidation of phenolic compounds in the presence of chemical additives has been the focus of this study. In this investigation, the influence of the additives polyethylene glycol and Triton X-100 was evaluated in the phenol oxidation, caffeic acid, chlorogenic acid and total phenolic compounds present in coffee processing wastewater (CPW) at different pH values, performed by turnip peroxidase and peroxidase extracted from soybean seed hulls. The influence of these additives was observed only in the oxidation of phenol and caffeic acid. In the oxidation of other studied phenolic compounds, the percentage of oxidation remained unchanged in the presence of these chemical additives. In the oxidation of CPW in the presence of additives, no change in the oxidation of phenolic compounds was observed. Although several studies show the importance of evaluating the influence of additives on the behaviour of enzymes, this study found a positive response from the economic point of view for the treatment of real wastewater, since the addition of these substances showed no influence on the oxidation of phenolic compounds, which makes the process less costly.

To obtain lactic acid bacteria that scavenge environmental hydrogen peroxide, we developed a specialized enrichment medium and successfully isolated Pediococcus pentosaceus Be1 strain from a fermented food. This strain showed vigorous environmental hydrogen peroxide scavenging activity over a wide range of hydrogen peroxide concentrations. High Mn-catalase and NADH peroxidase activities were found in the cell-free extract of the P. pentosaceus Be1 strain, and these two hydrogen peroxide scavenging enzymes were purified from the cell-free extract of the strain. Mn-catalase has been purified from several microorganisms by several researchers, and the NADH peroxidase was first purified from the original strain in this report. After cloning the genes of the Mn-catalase and the NADH peroxidase, the deduced amino acid sequences were compared with those of known related enzymes.

Background: In view of compliance with increasingly stringent environmental legislation, an eco-friendly treatment technology of industrial dyes and effluents is a major environmental challenge in the color industry. In present study, a promising and eco-friendly entrapment approach was adopted to immobilize purified manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum IBL-05 on Ca-alginate beads. The immobilized MnP was subsequently used for enhanced decolorization and detoxification of textile reactive dyes)., Results: MnP isolated from solid-state culture of G. lucidum IBL-05, presented highest immobilization yield (83.9 %) using alginate beads prepared at optimized conditions of 4 % (w/v) sodium alginate, 2 % (w/v) Calcium chloride (CaCl2) and 0.5 mg/ml enzyme concentration. Immobilization of MnP enhanced optimum temperature but caused acidic shift in optimum pH of the enzyme. The immobilized MnP showed optimum activity at pH 4.0 and 60 °C as compared to pH 5.0 and 35 °C for free enzyme. The kinetic parameters K(m) and V(max) of MnP were significantly improved by immobilization. The enhanced catalytic potential of immobilized MnP led to 87.5 %, 82.1 %, 89.4 %, 95.7 % and 83 % decolorization of Sandal-fix Red C4BLN, Sandal-fix Turq Blue GWF, Sandal-fix Foron Blue E2BLN, Sandal-fix Black CKF and Sandal-fix Golden Yellow CRL dyes, respectively. The insolubilized MnP was reusable for 7 repeated cycles in dye color removal. Furthermore, immobilized MnP also caused a significant reduction in biochemical oxygen demand (BOD) (94.61-95.47 %), chemical oxygen demand (COD) (91.18-94.85 %), and total organic carbon (TOC) (89.58-95 %) of aqueous dye solutions., Conclusions: G. lucidum MnP was immobilized in Ca-alginate beads by entrapment method to improve its practical effectiveness. Ca-alginate bound MnP was catalytically more vigorous, thermo-stable, reusable and worked over wider ranges of pH and temperature as compared to its free counterpart. Results of cytotoxicity like hemolytic and brine shrimp lethality tests suggested that Ca-alginate immobilized MnP may effectively be used for detoxification of dyes and industrial effluents.

White-rot fungi are the only organisms known to degrade all basic wood polymers using different strategies of employing a variety of hydrolytic and oxidative enzymes. A comparative secretome analysis of Termitomyces sp. OE147 cultivated on cellulose and lactose was carried out by two-dimensional gel electrophoresis followed by MALDI-TOF/TOF-MS analysis to identify the enzymes coordinately expressed on cellulose. A total of 29 proteins, belonging to CAZy hydrolases (11), CAZy oxidoreductases (13) and some 'other' (5) proteins were identified. Among the CAZy hydrolases, a distinct repertoire of cellulolytic and hemicellulolytic enzymes were produced while among the CAZy oxidoreductases, cellobiose dehydrogenase and laccase were the predominant enzymes along with H2O2 dependent peroxidases. This coordinated expression indicated a unique and integrated system for degradation of not only crystalline cellulose but also other components of lignocellulolytic substrates, namely lignin and xylan. Activities of the identified proteins were confirmed by plate assays and activity measurements. Many of the enzyme activities were also correlated with reduction in the crystallinity index of cellulose. Based on the enhanced production of CDH, β-glucosidases and several oxidoreductases, a more prominent role of these enzymes is indicated in this fungus in cellulose breakdown., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 μg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 μM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture)., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Magnetosomes are intracellular structures produced by magnetotactic bacteria and are magnetic nanoparticles surrounded by a lipid bilayer membrane. Magnetosomes reportedly possess intrinsic enzyme mimetic activity similar to that found in horseradish peroxidase (HRP) and can scavenge reactive oxygen species depending on peroxidase activity. Our previous study has demonstrated the phototaxis characteristics of Magnetospirillum magneticum strain AMB-1 cells, but the mechanism is not well understood. Therefore, we studied the relationship between visible-light irradiation and peroxidase-like activity of magnetosomes extracted from M. magneticum strain AMB-1. We then compared this characteristic with that of HRP, iron ions, and naked magnetosomes using 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in the presence of H2O2. Results showed that HRP and iron ions had different activities from those of magnetosomes and naked magnetosomes when exposed to visible-light irradiation. Magnetosomes and naked magnetosomes had enhanced peroxidase-like activities under visible-light irradiation, but magnetosomes showed less affinity toward substrates than naked magnetosomes under visible-light irradiation. These results suggested that the peroxidase-like activity of magnetosomes may follow an ordered ternary mechanism rather than a ping-pong mechanism. This finding may provide new insight into the function of magnetosomes in the phototaxis in magnetotactic bacteria., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Coculturing of two white-rot fungi, Dichomitus squalens and Ceriporiopsis subvermispora, was explored for the optimization of cultivation media for simultaneous augmentation of laccase and peroxidase activities by response surface methodology (RSM). Nutrient parameters chosen from our previous studies with the monocultures of D. squalens and C. subvermispora were used to design the experiments for the cocultivation study. Glucose, arabinose, sodium nitrate, casein, copper sulfate (CuSO4 ), and manganese sulfate (MnSO4 ) were combined according to central composite design and used as the incubation medium for the cocultivation. The interaction of glucose and sodium nitrate resulted in laccase and peroxidase activities of approximately 800 U/g protein. The addition of either glucose or sodium nitrate to the medium also modifies the impact of other nutrients on the ligninolytic activity. Both enzyme activities were cross-regulated by arabinose, casein, CuSO4 , and MnSO4 as a function of concentrations. Based on RSM, the optimum nutrient levels are 1% glucose, 0.1% arabinose, 20 mM sodium nitrate, 0.27% casein, 0.31 mM CuSO4 , and 0.07 mM MnSO4 . Cocultivation resulted in the production of laccase of 1,378 U/g protein and peroxidase of 1,372 U/g protein. Lignin (16.9%) in wheat straw was degraded by the optimized enzyme mixture., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

Alkylhydroperoxide reductase (AhpC, the 26 kDa antigen) is one of the abundant antioxidant enzymes in Helicobacter pylori and seems to have a good potential for use in development of immunoassays to detect H. pylori infection in clinical specimens. This study aimed to investigate some properties of this antigen by the produced monoclonal antibodies. Five established hybridoma cell lines secreting monoclonal antibodies (MAbs) against 26 kDa antigen of H. pylori were cultivated and MAbs were purified by affinity chromatography. Subsequently, MAbs were conjugated with biotin, and different combinations of capture and tracer antibodies used in sandwich ELISA. Immunoblotting of bacterial extracts were performed to estimate aggregation status of the antigen. Release of antigen from the cultivated bacteria on solid media was examined by sandwich ELISA, and also, existence of interference in fecal extract was investigated by immunoblotting and sandwich ELISA. Our findings showed that the MAbs against 26 kDa antigen of H. pylori could recognize three bands of nearly 25 kDa, 50 kDa, and 75 kDa in immunoblotting. This study also indicated presence of more antigens in the culture medium around the bacteria than the bacterial extract itself. The results of sandwich ELISA and immunoblotting on fecal extracts suggest the presence of interfering agents that prevent detection of antigen by antibody in ELISA but not in immunoblotting. In this study the oligomerization of the 26 kDa antigen, presence of interfering agents in stool matrix, and release of antigen to outside of bacteria, were demonstrated.

In silico analysis together with cloning, molecular characterization and heterologous expression reports that the hypothetical protein All5371 of Anabaena sp. PCC7120 is a novel hydroperoxide scavenging protein similar to AhpD of bacteria. The presence of E(X)11CX HC(X)3H motif in All5371 confers peroxidase activity and closeness to bacterial AhpD which is also reflected by its highest 3D structure homology with Rhodospirillum rubrum AhpD. Heterologous expression of all5371 complimented for ahpC and conferred resistance in MJF178 strain (ahpCF::Km) of Escherichia coli. All5371 reduced the organic peroxide more efficiently than inorganic peroxide and the recombinant E. coli strain following exposure to H2O2, CdCl2, CuCl2, heat, UV-B and carbofuron registered increased growth over wild-type and mutant E. coli transformed with empty vector. Appreciable expression of all5371 in Anabaena sp. PCC7120 as measured by qRT-PCR under selected stresses and their tolerance against H2O2, tBOOH, CuOOH and menadione attested its role in stress tolerance. In view of the above, All5371 of Anabaena PCC7120 emerged as a new hydroperoxide detoxifying protein.

This work focused on photo-assisted crude peroxidase mediated transformations of chlorinated phenols (CPs) from spiked and industrial wastewaters and the identification of reaction products formed. Garden radish Raphanus sativus was the source of crude peroxidase. No chlorine bearing compounds were detected by gas chromatography-high resolution mass spectrometry analysis. Under identical test conditions, the concentrations of 4-chlorophenol and 2,4-dichlorophenol were demoted to zero from 514 mg/L, 652 mg/L and that of 2,4,6-trichlorophenol and pentachlorophenol were reduced to 18 mg/L and 37 mg/L from 790 mg/L and 1066 mg/L, respectively (high-pressure liquid chromatography analysis). Chloride ion release profiles also showed a progressively increasing trend. A neat chemical oxygen demand removal to the extent of 63-79% was achieved in the case of spiked wastewater sample and to the extent of 77% for industrial wastewaters. A hypothesis reaction scheme was also suggested to comprehend the mechanism of degradation reactions.

Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn(2+) to Mn(3+), which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn(2+) binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn(2+) oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hydroxylated polybrominated diphenyl ethers (HO-PBDEs) are emerging endocrine-disrupting compounds that are widely present in the marine environment. The origin of HO-PBDEs is generally attributed to metabolism of PBDEs and natural production in the environment. However, it is unclear how HO-PBDEs are produced naturally. Here we report the formation of HO-PBDEs from simple bromophenols (BPs) [e.g., 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP)] under the catalysis of bromoperoxidase (BPO) isolated from the common marine red alga Corallina officinalis. Experiments at room temperature showed that BPO readily catalyzes the conversion of 2,4-DBP and 2,4,6-TBP to HO-PBDEs in the presence of Br(-) and H2O2. From analysis of the original forms and their corresponding methylated derivatives, the reaction products were tentatively identified as 2'-HO-BDE-121 and 4'-HO-BDE-121. The formation of HO-PBDEs was likely resulted from the coupling of bromophenoxy radicals generated by the oxidation of BPs via BPO-mediated processes. The presence of Br(-) in the reaction favored the conversion. The production of HO-PBDEs was found to be pH-dependent, and a higher yield was obtained at pH 6.5. In view of the abundance of BPs and C. officinalis in the marine environment, bioconversion of BPs mediated by BPO may be a potential route for the natural production of HO-PBDEs.

Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters.

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.

Background: Avocado (Persea americana Mill, cv. Hass) fruit ranks tenth in terms of the most important products for Mexico. Avocado products are quite unstable due to the presence of oxidative enzymes such as polyphenol oxidase and peroxidase. The present study is to characterize the activity of purified avocado peroxidase from avocado in order to ascertain the biochemical and kinetic properties and their inhibition conditions., Results: Purification was performed by Sephacryl S 200 HR gel filtration chromatography and its estimated molecular weight was 40 kDa. The zymogram showed an isoelectric point of 4.7. Six substrates were tested in order to ascertain the affinity of the enzyme for these substrates. The purified peroxidase was found to have low Km (0.296 mM) and high catalytic efficiency (2688 mM(-1) s(-1)) using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), optimum activity being reached at 51°C, pH 3.8. The addition of dithiothreitol, β-mercaptoethanol, ascorbic acid, sodium azide, L-cysteine and Tween-20 had high inhibitory effects, while metals ions such as Cu(+), Fe(2+) and Mn(2+) had weak inhibitory activity on purified avocado peroxidase., Conclusion: The purified avocado peroxidase exhibits high inhibition (Ki = 0.37 µM) with 1.97 µM n-propyl gallate using ABTS as substrate at 51°C, pH 3.8 for 10 min., (© 2013 Society of Chemical Industry.)

Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81 U mL(-1), specific activity 78 U mg(-1) with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4-5 and the optimum temperature was 25 °C. The pure MnP activity was enhanced by Mn(2+), Cu(2+), Ca(2+) and K(+) and inhibited by Hg(+2) and Cd(+2). H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL(-1) enzyme activities.

A cell-free protein synthesis system can produce various types of proteins directly from DNA templates such as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-throughput functional screening of proteins. Here, we report successful expression of active Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia coli cell-free protein synthesis system, wherein reaction conditions such as the concentrations of hemin, calcium ions, and disulfide bond isomerase were optimized to increase the solubility and activity of the synthesized enzyme. Moreover, cell-free synthesized MnP purified using the hemagglutinin tag showed higher specific activity than the commercial wild-type enzyme, suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide bond-containing metalloenzymes such as MnP. We believe that our system is a solid foundation for the development of a high-throughput screening method for the directed evolution of these enzymes., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

Dye-decolourizing peroxidases are haem-containing peroxidases with broad substrate specificity. Using H2O2 as an electron acceptor, they efficiently decolourize various dyes that are of industrial and environmental relevance, such as anthraquninone- and azo-based dyes. In this study, the dye-decolourizing peroxidase DdDyP from Dictyostelium discoideum was overexpressed in Escherichia coli strain Rosetta(DE3)pLysS, purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.65 Å resolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 141.03, c = 95.56 Å, α = β = γ = 90°. The asymmetric unit contains two molecules.