Extracellular Peroxidase Activity Research Articles

Down-regulation of pyruvate decarboxylase gene of white-rot fungus Phlebia sp. MG-60 modify the metabolism of sugars and productivity of extracellular peroxidase activity

Ethanologenic white-rot fungus Phlebia sp. MG-60-P2 produces ethanol directly from several lignocelluloses. Efficient gene silencing methods are needed for metabolic engineering of this fungus for biorefinery use. In this study, we evaluated the effectiveness of RNAi-mediated silencing of the pyruvate decarboxylase gene of Phlebia sp. MG-60-P2 (MGpdc1). The RNAi lines generated showed a variety of suppression levels of ethanol production and MGpdc1 expression, and two selected strains led to different metabolic fluxes, resulting in rapid accumulation of xylitol from xylose. Knockdown lines KD2 and KD10 showed different strength of silencing. The moderate-inhibition line (KD10) showed faster xylitol accumulation from xylose than the severe-inhibition line (KD2). KD2, KD10 and knockout line KO77 showed higher extracellular peroxidase activity than the wild-type. Gene silencing using RNAi for MGpdc1 in the ethanologenic white-rot fungus Phlebia sp. MG-60-P2 is an effective first step in metabolic engineering to produce other chemicals besides ethanol. This high efficiency of transformation and silencing effect will make it possible to cotransform with multiple expression vectors which enhance the minor metabolic pathway or introduce exogenous metabolic reaction in Phlebia sp. MG-60-P2.

Changes in apoplastic peroxidase activity and cell wall composition are associated with cold-induced morpho-anatomical plasticity of wheat leaves.

Temperate grasses, such as wheat, become compact plants with small thick leaves after exposure to low temperature. These responses are associated with cold hardiness, but their underlying mechanisms remain largely unknown. Here we analyse the effects of low temperature on leaf morpho-anatomical structure, cell wall composition and activity of extracellular peroxidases, which play key roles in cell elongation and cell wall thickening, in two wheat cultivars with contrasting cold-hardening ability. A combined microscopy and biochemical approach was applied to study actively growing leaves of winter (ProINTA-Pincén) and spring (Buck-Patacón) wheat developed under constant warm (25°C) or cool (5°C) temperature. Cold-grown plants had shorter leaves but longer inter-stomatal epidermal cells than warm-grown plants. They had thicker walls in metaxylem vessels and mestome sheath cells, paralleled with accumulation of wall components, predominantly hemicellulose. These effects were more pronounced in the winter cultivar (Pincén). Cold also induced a sharp decrease in apoplastic peroxidase activity within the leaf elongating zone of Pincén, and a three-fold increase in the distal mature zone of the leaf. This was consistent with the enhanced cell length and thicker cell walls in this cultivar at 5°C. The different response to low temperature of apoplastic peroxidase activity and hemicellulose between leaf zones and cultivar types suggests they might play a central role in the development of cold-induced compact morphology and cold hardening. New insights are presented on the potential temperature-driven role of peroxidases and hemicellulose in cell wall dynamics of grasses.

Extracellular Peroxidase Activity and Light Emission of the Mycelium of the Basidiomycete Neonothopanus nambi in the Presence of β-Glucosidase

A comparative evaluation of the level of extracellular peroxidase activity and light-emission intensity of the mycelium of the luminescent basidiomycete Neonothopanus nambi in the presence of β-glucosidase was performed. The enzyme activity damages the hyphae of the fungus leading to osmotic imbalance, partial degradation of the mycelium, and release of extracellular peroxidases into the incubation medium. The presence of β-glucosidase reduces the time necessary to reach the maximum luminescence. Putative biochemical mechanisms that underlie the stimulation of reactive oxygen species formation (first and foremost, of hydrogen peroxide) in the N. nambi mycelium in the presence of β-glucosidase are proposed.

Estimating levels of light emission and extracellular peroxidase activity of mycelium of luminous fungus Neonothopanus nambi treated with β-glucosidase

Estimating levels of light emission and extracellular peroxidase activity of mycelium of luminous fungus Neonothopanus nambi treated with β-glucosidase

Functional characterization of two bZIP transcription factors in Verticillium dahliae

bZIP transcription factors play various biological roles in stress responses, conidiation, and pathogenicity in pathogenic fungi. Here, we report two bZIP transcription factors (VDAG_08640 and VDAG_08676) of Verticillium dahliae, which were differentially expressed during microsclerotia development and induced by hydrogen peroxide as well. We find that deletion of either gene does not affect microsclerotia formation and the sensitivity to hydrogen peroxide; however, the mutants manifest decreased activity of extracellular peroxidase and laccase. Other phenotypic characterization reveals that VDAG_08676 disruption results in significant reduction of conidial production and virulence, while VDAG_08640 disruption does not lead to observable phenotypic variances compared with the wild-type strain. To elucidate whether they exhibit functional redundancy, double deletion mutants were generated. The double deletion mutants show remarkably increased sensitivity to hydrogen peroxide stress, whereas the two genes are not involved in microsclerotia formation. Taken together, our data demonstrate that a bZIP transcription factor gene VDAG_08676 is involved in the conidial production, oxidative stress response and virulence which may lay a foundation for further analysis of other bZIP transcription factors in V. dahliae.

MoVrp1, a putative verprolin protein, is required for asexual development and infection in the rice blast fungus Magnaporthe oryzae

Endocytosis is a crucial cellular process in eukaryotic cells which involves clathrin and/or adaptor proteins, lipid kinases, phosphatases and the actin cytoskeleton. Verprolin proteins, such as Vrp1 in Saccharomyces cerevisiae, are conserved family proteins that regulate actin binding and endocytosis. Here, we identified and characterized MoVrp1 as the yeast Vrp1 homolog in Magnaporthe oryzae. Deletion of the MoVRP1 gene resulted in defects in vegetative growth, asexual development, and infection of the host plant. The ∆Movrp1 mutants also exhibited decreased extracellular peroxidase and laccase activities and showed defects in colony pigmentation, hyphal surface hydrophobicity, cell wall integrity, autophagy, endocytosis, and secretion of avirulent effector. Our studies provided new evidences that MoVrp1 involved in actin cytoskeleton is important for growth, morphogenesis, cellular trafficking, and fungal pathogenesis.

Morphological properties and levels of extracellular peroxidase activity and light emission of the basidiomycete Armillaria borealis treated with β-glucosidase and chitinase

Morphological properties and levels of extracellular peroxidase activity and light emission of the basidiomycete Armillaria borealis treated with β-glucosidase and chitinase

Using aquatic fungi for pharmaceutical bioremediation: Uptake of acetaminophen by Mucor hiemalis does not result in an enzymatic oxidative stress response

The increasing anthropogenic pollution of aquatic environments and fresh water scarcity worldwide have prompted the development of low-cost and effective water treatment alternatives. One example of a highly released anthropogenic xenobiotics is acetaminophen (APAP), which has been detected in surface waters at concentrations as high as 5 μg L−1. To date, traditional water treatment plants were unable to remove all pharmaceutical xenobiotics and as in the case with APAP, the breakdown products are toxic. Phytoremediation has proved to remove xenobiotics efficiently producing no toxic breakdown products, however, they are often restrained in their application range. Therefore, it was necessary to find alternate remediation tools to extend and complement the application ranges of existing bioremediation techniques. With the success of mycoremediation as well as the adaptability of fungi, Mucor hiemalis was investigated in terms of its APAP uptake capabilities. The investigation included the examination of concentration- and time-dependent uptake studies to examine the effects of each of these parameters independently. Additionally, the extracellular peroxidase activity of M. hiemalis was measured with exposure to APAP to evaluate possible breakdown and the antioxidative stress enzymes, catalase, glutathione peroxidase, and glutathione reductase, were assayed to investigate whether APAP caused oxidative stress. The results showed that M. hiemalis was able to internalize between 1 and 2 μg APAP per g dried fungal biomass when exposed to 5, 10, 50 and 100 ng mL−1 APAP for 24–48 h, but not beyond this time frame. Further, exposure to APAP did not result in elevated extracellular peroxidase activity or oxidative stress. The findings led to the conclusion that M. hiemalis could be integrated in bioremediation systems, for short-term degradation at low concentrations of APAP with effective management.

Effect of iron on the probiotic properties of the vaginal isolate Lactobacillus jensenii CECT 4306.

The vaginal microbiota of healthy, fertile women is dominated by lactobacilli. As a defence mechanism, these bacteria produce H₂O₂ to discourage colonization of the vagina by undesirable micro-organisms. In particular, Lactobacillus jensenii CECT 4306 is a strong producer of H₂O₂ and has been found to protect itself from the bactericidal effects of this compound through the activity of extracellular peroxidases. However, this peroxidase activity is dependent on the presence of Fe(3+), which is found in elevated concentrations in the vaginal mucosa as a consequence of the menstrual discharge. The aim of the present work was to evaluate whether Fe(3+) is able to modulate other potential probiotic properties of strain 4306. We found that Fe(3+) enhances the adhesion of L. jensenii CECT 4306 to mucin and to HT-29 and HT-29 MTX cells, and, in addition, improves the anti-inflammatory profile, as judged by an increase in the ratio of IL-10/IL-12p70 that were secreted by macrophages. A comparison of total, secreted and surface proteins produced in the presence and absence of Fe(3+) revealed significant differences in the concentration of the moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In conclusion, Fe(3+) seems to improve the probiotic characteristics of L. jensenii CECT 4306, and future research of the interactions of this strain with its vaginal environment may reveal further information about different aspects of its probiotic potential.

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

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

Screening of microscopic fungi and their enzyme activities for decolorization and biotransformation of some aromatic compounds

A total of 610 strains from 16 species of micromycetes with potential ligninolytic capabilities were isolated from soil, compost, and rotten wood, including 400 strains of soil origin. In the test of decolorization of agarized 0.06 % alizarin blue, 52 strains with high decolorization activity were selected. In liquid cultures with 0.2 % post-industrial lignin and 0.03 % alizarin blue, all of the selected strains showed three different extracellular peroxidase activities such as HRP-like, LiP, and MnP. Laccase was detected only in the cultures with alizarin blue, but those were trace amounts. In the decolorization of alizarin blue, the highest levels of activity were attained by HRP-like peroxidase. In the transformations of post-industrial lignin that included decolorization, colorization (darkening of substrate), and re-colorization, the highest levels of activity were attained by lignin peroxidase, especially at the start of the culture. The transformations of both dye substrates in the cultures of all 52 strains were accompanied by the release of glucose oxidase, but high activities of that enzyme were observed only in the presence of alizarin blue. Whereas in the presence of lignin the fungi under study synthesized catalase, the activity of which attained a maximum toward the end of the culture was negatively correlated with the activity of the remaining oxidoreductases under study. Among the 52 selected strains, three strains such as Haematonectria haematacocca BwIII43, K37, and Trichoderma harzianum BsIII33 were characterized by a high activity of decolorization.

Growth and oxidative enzymatic activity of in-vitro cultures of Ciliochorella buxifolia

To get a better insight into the physiological capabilities of Ciliochorella buxifolia, the most frequent fungus occurring on Scutia buxifolia leaf-litter in a native forest from Argentina, its in-vitro ability to use 10 carbon sources and to produce extracellular enzymes, including its response to tannic acid and to the addition of a water-soluble fraction of Scutia buxifolia leaf-litter, was analyzed. Growth, colony morphology and extracellular enzyme activity as well as differentiation of pycnidia were a function of the C substrate. The fungus responded to the presence of tannic acid in a range between 0.001 and 0.1% (w/v), by increasing growth, but higher phenol concentrations like 0.5% were inhibitory. The activity of extracellular oxidative enzymes increased with the concentration of tannic acid. Furthermore, the fungus showed extracellular laccase and peroxidase activity, being the former increased by water-soluble fraction in association to pycnidia development. Based on these results, Ciliochorella buxifolia is a fungus growing on Scutia buxifolia leaf-litter that is able to metabolize soluble phenolic compounds, which triggers the synthesis of extracellular oxidative enzymes possibly involved in sporulation and detoxification reactions.

Effect of jasmonic acid on the pro-/antioxidant system of wheat coleoptiles as related to hyperthermia tolerance

The effects of treatment with jasmonic acid (JA) of wheat (Triticum aestivum L, cv. Elegia) coleoptiles on the generation of superoxide anion-radical (O2·−), the activity of extracellular peroxidase, enzymatic and non-enzymatic components of the antioxidant system were studied. During the first hour after the start of coleoptile treatment with 1 μM JA, the generation of O2·− was enhanced and the extracellular peroxidase was activated. During following 23 h, these effects were gradually reduced. JA-enhanced O2·− generation was partially suppressed by coleoptile treatment with the inhibitor of peroxidase salicylhydroxamic acid, the inhibitor of NADPH-oxidase imidazol, and also the calcium chelator EGTA. Under the influence of JA treatment, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, and soluble guaiacol peroxidase) in wheat coleoptiles were activated. Treatment with JA improved coleoptile tolerance to damaging heating (10 min at 43°C); it favored the maintenance of the pools of enzymatic and non-enzymatic antioxidants. The inhibitors of NADPH-oxidase and peroxidase, and also calcium chelator reduced a positive JA influence on coleoptile thermotolerance. The role of changes in the pro-/antioxidant balance in plant tissues for the realization of stress-defensive JA effects is discussed.

Biochemical and molecular characterization of an atypical manganese peroxidase of the litter-decomposing fungus Agrocybe praecox

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 Mn2+ to Mn3+, 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 Mn2+ 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 Mn2+ oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer.

A correlation between the fate and non-extractable residue formation of 14C-metalaxyl and enzymatic activities in soil

Extracellular, oxidative soil enzymes like monophenol oxidases and peroxidases play an important role in transformation of xenobiotics and the formation of organic matter in soil. Additionally, these enzymes may be involved in the formation of non-extractable residues (NERs) of xenobiotics during humification processes. To examine this correlation, the fate of the fungicide 14C metalaxyl in soil samples from Ultuna (Sweden) was studied. Using different soil sterilization techniques, it was possible to differentiate between free, immobilized, and abiotic (“pseudoenzyme”-like) oxidative activities. A correlation between the formation of metalaxyl NER and soil organic matter content, biotic activities, as well as extracellular phenoloxidase and peroxidase activities in the bulk soil and its particle size fractions was determined. Extracellular soil-bound enzymes were involved in NER formation (up to 8% of applied radioactivity after 92 days) of the fungicide independently from the presence of living microbes and different distributions of the NER in the soil humic subfractions.

Selenium pharmaconutrition in sepsis: To give or not to give? Is this still the question?

The Surviving Sepsis Campaign (SSC) International Guidelines for Management of Severe Sepsis and Septic Shock [1] were recently updated to assist critical care practitioners in managing the proliferation of emerging evidence for the treatment of sepsis syndromes and improving the prognosis of sepsis. Regarding pharmaconutrition with high-dose intravenous (IV) selenium, we find it surprising that these new SSC guidelines suggest, “not using intravenous selenium for the treatment of severe sepsis (grade 2C)” [1]. We respectfully disagree with this recommendation and question the rationale for this conclusion. In our opinion, the SSC may not have considered much of the published clinical evidence. In fairness, the author’s recommendation does not exclude the use of low-dose selenium in parenteral nutrition (PN) but no reference is made to the American Society for Parenteral and Enteral Nutrition (ASPEN) position paper on micronutrients [2] indicating that, although controversial, “patients who are deficient in selenium or who are critically ill, septic, or have severe burns may benefit from short termvery high daily doses [of selenium] administered separately from the PN” [2]. In fact, several observational studies have demonstrated that: low selenium status is associated with severity of illness; septic shock patients exhibit the lowest plasma selenium levels; and serum selenium shows a relatively good predictive value for mortality in the intensive care unit (ICU) [3–5]. Therefore, it seems logical that selenium supplementation is not only able to correct a nutritional deficiency but can help to improve outcome for septic patients. One of the most relevant selenium pharmaconutrition trials, quoted by the SSC was the SIC (Selenium in Intensive Care) study [6]. The SIC protocol incorporated an initial selenite loading dose by IV bolus to optimize selenium levels and extracellular glutathione peroxidase activity, followed by continuous infusion for 14 d. Although the reduction in 28-d mortality for supplemented patients failed to reach statistical significance in the intentionto-treat analysis, mortality was significantly lower in the perprotocol group (56.7% versus 42.6%; P 1⁄4 0.049). Additionally, mortality was significantly reduced in those septic patients with

Agaricus bisporus and related Agaricus species on lignocellulose: Production of manganese peroxidase and multicopper oxidases

Biotechnological, microbiological, and genetic studies of Agaricus species other than A. bisporus, the white button mushroom, have been limited so far. To expand the knowledge in the genus Agaricus, six novel wild-type isolates of Agaricus spp. were studied on their nutritional demands for enzyme production and mycelial growth. All the selected Agaricus species produced extracellular manganese peroxidase (MnP) and laccase activities in semi-solid rye bran cultures. Moderate MnP activities were measured for A. bisporus, A. bernardii and A. campestris. The highest laccase activities were obtained for A. bisporus and A. campestris. On soy medium, the highest mycelial tyrosinase activity was determined for A. bernardii. For A. bisporus, addition of copper caused no increase in laccase or tyrosinase activities on soy or malt extract media. Hyphal growth rate of the isolates was studied on lignocellulose amended agar plates. Fastest growth was obtained for A. bisporus on wheat bran and birch leaf litter agar. Except for A. bernardii, hyphal growth rates correlated well with MnP and laccase production levels between Agaricus species. Molecular taxonomy of the novel Agaricus spp. positioned them to distinct phylogenetic clusters with species-level identity. In conclusion, our data point to the importance of both MnP and multicopper enzymes in Agaricus spp. while growing on lignocelluloses.

Effect of vitamin E on the immune system of ewes during late pregnancy and lactation

The present experiment was designed to determine the effects of a regimen of repeated, intramuscular (i.m.) injections of vitamin E (VE) on innate and humoral components of the immune response of pregnant and lactating ewes. Pregnant ewes were randomly assigned to two treatments consisting of i.m. injections of either VE (30IU d-α-tocopherol/kg BW, n=10) or equivalent volume of a placebo (emulsified base, n=8). Injections were administered at 2-week (wk) intervals to all ewes, as a group, for a total of 24wk. Lambing occurred over a 5-wk period, starting wk 7 of the study. All ewes were administered a booster vaccination for Clostridium tetani and Clostridium perfringens (C+D) at wk 3. Blood and colostrum were analyzed for α-tocopherol and selenium content, and extracellular glutathione peroxidase (GPX3) activity as measures of antioxidant status; lysozyme activity as a measure of innate immunity and immunoglobulin G (IgG), and anti-tetanus toxoid IgG (IgG-T) concentration as a measure of humoral immune status. Three ewes in the placebo group did not lamb and were therefore excluded from all analyses. Results showed a significant treatment×week effect of VE supplementation on IgG concentration. VE supplementation increased IgG concentrations in ewes after their yearly booster vaccination against C. tetani and C. perfringens; placebo-treated ewes showed no such response. The extent, magnitude and persistence of the decreased IgG concentration during the transition period were less in VE-supplemented ewes than controls. There was no effect of VE supplementation on the concentration of IgG-T after vaccination. VE-supplementation of pregnant ewes had no effect on serum lysozyme activity. GPX3 increased in both treatment groups, peaking during the transition period. The α-tocopherol content of the colostrum of VE-supplemented ewes was greater than that of controls. In conclusion, VE supplementation of pregnant and lactating ewes had an equivocal effect on variables used to assess immune function. The benefit of VE supplementation to pregnant and lactating ewes requires further research however it may have utility in blunting the immune suppression that occurs during the transition period.

A two-component histidine kinase, MoSLN1, is required for cell wall integrity and pathogenicity of the rice blast fungus, Magnaporthe oryzae

A two-component signal transduction system is a common mechanism for environmental sensing in bacteria. The functions of the two-component molecules have been also well characterized in the lower eukaryotic fungi in recent years. In Saccharomyces cerevisiae, the histidine kinase Sln1p is a major component of the two-component signaling pathways and a key regulator of the osmolarity response. To determine the function of MoSLN1, a Sln1 homolog of Magnaporthe oryzae, we cloned the MoSLN1 gene and generated specific mutants using gene knock-out strategy. Disruption of MoSLN1 resulted in hypersensitivity to various stresses, reduced sensitivity to cell wall perturbing agent Calcofluor white, and loss of pathogenicity, mainly due to a penetration defect. Additionally, we showed that MoSLN1 is involved in oxidative signaling through modulation of intra- and extracellular peroxidase activities. These results indicate that MoSLN1 functions as a pathogenicity factor that plays a role in responses to osmotic stress, the cell wall integrity, and the activity of peroxidases.

2,4,6-Trichlorophenol mediated increases in extracellular peroxidase activity in three species of Lemnaceae

Chlorinated phenols, or chlorophenols, are persistent priority pollutants that are widespread in the environment. Class III peroxidases are well-characterised plant enzymes that can catalyse the oxidative dechlorination of chlorophenols. Expression of these enzymes by plants is commonly associated with plant stress, therefore limiting scope for phytoremediation. In this study, we have quantitatively compared peroxidase activity and phytotoxicity as a function of 2,4,6-trichlorophenol (TCP) concentration in three species of Lemnaceae; Lemna minor, Lemna gibba and Landoltia punctata. Effects of TCP on the growth rates of the three species differed considerably with L. punctata being the most tolerant species. TCP also affected photosynthetic parameters, causing a decrease in open photosystem II reaction centres (qP) and, in L. punctata only, a decrease in non-photochemical quenching (qN). In parallel, TCP exposure resulted in increased peroxidase activity in all three species. Peroxidase activity in L. minor and L. gibba displayed an inverse relationship with biomass accumulation, i.e. the more growth reduction the more peroxidase activity. In contrast, induction of peroxidase activity in L. punctata was bi-phasic, with a TCP-induced activity peak at concentrations that had no major effect on growth, and further induction under phytotoxic concentrations. The mechanism by which L. punctata recognises and responds to low concentrations of an anthropogenic compound, in the absence of wide-ranging stress, remains enigmatic. However, we conclude that this “window” of peroxidase production in the absence of major growth inhibition offers potential for the development of sustainable, peroxidise-mediated phytoremediation systems.