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

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

Function of hesperidin alleviating inflammation and oxidative stress responses in COPD mice might be related to SIRT1/PGC-1α/NF-κB signaling axis.

Purpose: Hesperidin has anti-inflammatory and anti-oxidant stress effects, but its functions in chronic obstructive pulmonary disease (COPD) remains unknown. This study analyzed the role of hesperidin in COPD mice, aiming to provide a basis for the hesperidin application.Materials and methods: Mice were injected with cigarette smoke extract (CSE) to construct COPD models and then treated with budesonide or hesperidin. Hematoxylin-eosin (HE) and TUNEL assays were used to observe the pathological changes and cell death of lung tissue. The levels of interleukin (IL)-6, IL-8, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in bronchoalveolar lavage fluid (BLAF), as well as myeloperoxidase (MPO) content in lung tissues were confirmed. The expression levels of SIRT1, PGC-1α, and p65 proteins were measured by western blotting (WB) analysis.Results: CSE induced inflammatory cell infiltration and cell death in the lung tissues of mice, whereas budesonide and hesperidin effectively alleviated these pathological changes. The levels of IL-6, IL-8, and MDA in BLAF and pulmonary MPO content in the COPD mice were effectively increased, while the levels of SOD and CAT in BLAF were decreased, which could be reversed by budesonide and hesperidin. Moreover, the addition of budesonide or hesperidin reliably accelerated the expression levels of PGC-1α and SIRT1 but suppressed the phosphorylation of p65 in COPD mice. In general, high-dose hesperidin had a stronger regulatory effect on COPD mice.Conclusions: Hesperidin alleviated inflammation and oxidative stress responses in CES-induced COPD mice, associated with SIRT1/PGC-1α/NF-κB signaling axis, which might become a new direction for COPD treatment.

Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique.

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives were synthesized and identified as new competitive POD inhibitors. Then, these derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold, 22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD), black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to purify all radish species POD enzymes in high purity and yield.

Efficacy of the RemoweLL cardiotomy reservoir for fat and leucocyte removal from shed mediastinal blood: a randomized controlled trial.

INTRODUCTION: Re-transfusion of lipid particles and activated leucocytes with shed mediastinal blood (SMB) can aggravate cardiopulmonary bypass-associated inflammation and increase the embolic load. This study evaluated the fat and leucocyte removal capacity of the RemoweLL cardiotomy reservoir. METHODS: Forty-five patients undergoing elective on-pump cardiac surgery were randomly allocated to filtration of SMB using the RemoweLL or the Admiral cardiotomy reservoir. The primary outcome was a drop in leucocytes and lipid particles obtained with the two filters. The effect of the filters on other blood cells and inflammatory mediators, such as myeloperoxidase (MPO), was also assessed. RESULTS: The RemoweLL cardiotomy filter removed 16.5% of the leucocytes (p<0.001) while no significant removal of leucocytes was observed with the Admiral (p=0.48). The percentage reductions in lipid particles were similar in the two groups (26% vs 23%, p=0.2). Both filters similarly affected the level of MPO (p=0.71). CONCLUSION: The RemoweLL filter more effectively removed leucocytes from SMB than the Admiral. It offered no advantage in terms of lipid particle clearance.

Biobleaching of industrial important dyes with peroxidase partially purified from garlic.

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and V max for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5-5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

Heterologous expression of the msp2 gene from Marasmius scorodonius.

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca(2+), and hemin.

A new peroxidase from garlic (Allium sativum) bulb: its use in H2O2 biosensing.

Peroxidase POX(1) isoenzyme was purified from garlic (Allium sativum L.) bulb by ammonium sulfate precipitation, gel filtration and anion-exchange chromatography. Native-PAGE profile showed two isoforms, designated POX(1A) and POX(1B). POX(1B) seems to be more attractive for biosensor design since its K(m) (app) for H(2)O(2) is lower than that of POX(1A). In addition to its storage and operational stability, POX(1B) was found to be highly heat-stable, since almost 70% of its activity was conserved at 60 degrees C, whereas full activity was retained at 50 and 40 degrees C for 40 min. The optimal pH was approx. 5 and the optimal temperature was 30 degrees C. Next, gelatin was used as a matrix for enzyme immobilization on a gold electrode surface and electrochemical measurements were performed by using cyclic voltammetry. POX(1B)-based electrodes show great potential for application in H(2)O(2) monitoring of biological samples.

Isolation and characterization of antioxidation enzymes from cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 5-l bioreactor.

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40 degrees C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60 degrees C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.

Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions.

An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively.

Characterization of peroxidase in buckwheat seed.

A peroxidase (POX)-containing fraction was purified from buckwheat seed. The POX consisted of two isozymes, POX I and POX II, that were purified 6.6- and 67.4-fold, respectively. Their molecular weights were estimated to be 46.1 kDa (POX I) and 58.1 kDa (POX II) by gel filtration. While POX I and II each oxidized quercetin, o-dianisidine, ascorbic acid and guaiacol, only POX II oxidized ABTS. Kinetic studies revealed that POX I and II had lower K(m) values for quercetin (0.071 and 0.028 mM), ABTS (0.016 mM for POX II) and ascorbic acid (0.043 and 0.029 mM) than for o-dianisidine (0.229 and 0.137 mM) and guaiacol (0.288 and 0 ). The optimum pHs of POX I and II for various substrates were almost the same, except for quercetin; pH 8.0 for POX I and pH 4.5 for II. Their optimal temperatures were 30 degrees C (POX I) and 10 degrees C (POX II), and POX I was more stable than POX II above 30 degrees C.

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes.

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3',4'-anhydrovinblastine: a specific role for a multifunctional enzyme.

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

A 37-kDa peroxidase secreted from liverworts in response to chemical stress.

A peroxidase was purified from the culture medium of a suspension culture of Marchantia polymorpha (liverwort) after treatment with bornyl acetate, which acts as a chemical stress agent to the cells. The peroxidase was characterised as a glycoprotein of molecular mass 37-kDa having a pl of about 10 and an optimal pH of 6.5. The peroxidase was thermally stable at 50 degrees C for up to 60 min. The partial amino acid sequence of the peroxidase was determined and found to be dissimilar to the amino acid sequences of other higher plant peroxidases. The oxidative polymerization of lunularin by this peroxidase was examined and the formation of a dimer, a trimer and a tetramer was demonstrated by negative ion Fast Atom Bombardment (FAB)-mass spectroscopy of the reaction products.

Purification and cloning of the salivary peroxidase/catechol oxidase of the mosquito Anopheles albimanus.

Salivary homogenates of the adult female mosquito Anopheles albimanus have been shown previously to contain a vasodilatory activity associated with a catechol oxidase/peroxidase activity. We have now purified the salivary peroxidase using high-performance liquid chromatography. The pure enzyme is able to relax rabbit aortic rings pre-constricted with norepinephrine. The peroxidase has a relative molecular mass of 66 907 as estimated by mass spectrometry. Amino-terminal sequencing allowed us to design oligonucleotide probes for isolation of cDNA clones derived from the salivary gland mRNA from female mosquitoes. The full sequence of the cDNA demonstrated homology between A. albimanus salivary peroxidase and several members of the myeloperoxidase gene family. A close comparison of A. albimanus salivary peroxidase with canine myeloperoxidase, for which the crystal structure is known, showed that all six disulfide bridges were conserved and demonstrated identity for all five residues associated with a Ca2+-binding site. In addition, 16 of 26 residues shown to be in close proximity to the heme moiety in the canine myeloperoxidase were identical. We conclude that the salivary peroxidase of A. albimanus belongs to the myeloperoxidase gene family. Other possible functions for this molecule in blood feeding are discussed.

Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies.

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya (wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3',4'-trihydroxychalcone and 4, 3',4'-trihydroxy-3-methoxychalcone to the corresponding 3, 3'-biflavanones, as mixtures of racemic and meso forms.

Antiinflammatory effects of human milk on chemically induced colitis in rats.

We examined the effects of a human milk diet on rats with chemical colitis induced with a 4% acetic acid enema. Colonic myeloperoxidase activity was used as a surrogate marker for neutrophil infiltration. Control rats fed rat chow had little colonic myeloperoxidase activity; geometric mean, 0.27 U/g of tissue. Rats with colitis fed rat chow had significantly increased colonic myeloperoxidase activity (geometric mean, 6.76 U/g, p < 0.01 versus no colitis), as did rats with colitis fed infant formula or Pedialyte (geometric mean, 6.92 and 8.13 U/g, respectively, both p < 0.01 versus no colitis). Animals with colitis fed human milk had significantly lower colonic myeloperoxidase activity (geometric mean, 2.34 U/g) than did animals with colitis fed either chow or infant formula (p < 0.001). Similar effects were seen in rats with colitis fed infant formula supplemented with recombinant human IL-1 receptor antagonist (geometric mean, 1.95 U/g). These data show that orally administered human milk has an antiinflammatory effect on chemically induced colitis in rats, which may be mediated in part by IL-1 receptor antagonist contained in human milk.

Purification and characterization of a novel class III peroxidase isoenzyme from tea leaves.

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 +/- 10, mean +/- SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm (epsilon = 115 mM-1 cm-1) and an A406/A280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 mumol min-1 mg-1), with a pH optimum in the range of 4.5 to 5.0. Substrate-specificity studies have revealed significant differences between TP and other class III peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin pi cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

Human milk peroxidase is derived from milk leukocytes.

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.