Role of chitin-specific peroxidase in wilt-resistant cotton

Chitin-specific isoforms of peroxidase with molecular weights 67 and 54.7 kDa that are involved in cotton wilt-resistance were obtained from sprouts of cotton variety AN-Bayaut-2 using chromatography. Electron microscopy showed that the morphology of the fungus V. dahliae changed under the influence of the chitin-specific isoforms of peroxidase. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 397–399, July–August, 2008     

Neutral lipids from seeds of Asteraceae plants

Lipids from seeds of three plants of the Asteraceae family, Cousinia franchetii, Arctium leiospermum, and Rhaponticum integrifolium, were studied. The principal constituents of lipids from the three plants were shown to be acylglycerides of ordinary fatty acids and oxygenated fatty acids using chemical and chromatographic analyses. The composition of the ordinary unoxidized and epoxy acids was determined by GC. Presented at the 7th International Symposium on the Chemistry of Natural Compounds, October 16–18, 2007, Tashkent, Uzbekistan __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 115–117, March–April, 2008.     

Enzymatic method for the determination of phenols with the use of peanut peroxidase

Cationic peanut peroxidase was used for the first time for the determination of phenols at a level of 0.5–10μM. The examined phenols were found to be inhibitors or second substrates of peanut peroxidase in the indicator reaction of the oxidation ofo-dianisidine by hydrogen peroxide. The effect of phenols on the rate of the indicator reaction depends on their redox properties. The data on the effects of phenols on the catalytic activities of peroxidases isolated from different sources (peanut, horseradish roots,Medicago sativa alfalfa cells, and the xylotrophic fungusPhellinius igniarius) were compared     

The special features of protein adsorption isotherms on silica adsorbents

The adsorption isotherms of hemoglobin, peroxidase, and β-galactosidase on silochrome and mesoporous and biporous silicas were comparatively studied. Adsorption developed in two stages, including fast “reversible” protein adsorption (equilibrium was reached in t ≤ 1–2 h) and a “slow stage” of irreversible binding in t ≫ 24 h (multipoint adsorption). The corresponding equilibrium constants were determined. The mechanism of unlimited linear association of peroxidase in the adsorption layer on the surface of silochrome was established.     

Bi-Enzyme Alcohol Biosensors Based on Genetically Engineered Alcohol Oxidase and Different Peroxidases

We report on the development of a bi-layer bi-enzyme biosensor architecture using different peroxidases and alcohol oxidase from Hansenula polymorpha C-105 as biological recognition elements. The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree peroxidase crosslinked with an Osmium complex-modified redox hydrogel. On top, a second layer was formed by electrochemically induced precipitation of a cathodic electrodeposition paint simultaneously entrapping alcohol oxidase isolated from a genetically modified strain of Hansenula polymorpha C-105. The sensor architecture was optimized with respect to effective electron transfer and stability of the enzyme. The main characteristics of the biosensors are an apparent maximal current I_max^app of 572–940 nA, an apparent Michaelis constant K_M^app of 9.5 mM, a sensitivity of 60–98 nA mM⁻¹ and an improved operational stability represented by a deactivation constant of 1.5–2.0 × 10⁻⁴min⁻¹.     

Selection and regeneration of groundnut plants resistant to the pathotoxic culture filtrate ofCercosporidium personation through tissue culture technology

Callus cultures were established from immature leaf expiants ofArachis hypogaea on MS medium supplemented with 2.0 mg/L of NAA and 0.5 mg/L of BAP of the susceptible cultivars namely VRI-2 and TMV-7. Three-week-old calli were subjected to mutagenic treatments (gamma rays: 50–250 Gy and EMS: 5–25 mM). Mutagen-treated calli were subcultured to fresh medium containing various concentrations (25–100% v/v) of pathotoxic culture filtrates. Calli were challenged in vitro with pathotoxic culture filtrate of the fungal pathogen and were assessed by visible growth ratings expressed as the percent response to the doses/concentrations of mutagen. Selected mutagen-treated calli showed resistance in vitro on media containingCercosporidium personatum pathotoxic culture filtrate. Resistance calli were then transferred to MS regeneration medium supplemented with BAP (2.0 mg/L) and NAA (0.5 mg/L) for shoot bud regeneration. The progeny of the plants produced 13 disease-resistant plants (R₂) in both the cultivars. Among the eight R₂ populations studied, 70.2–82.5% of the plants exhibited enhanced resistance. This study suggested that groundnut plants with resistance to C.personatum can be selected     

Degradation of textile dyes mediated by plant peroxidases

The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25–200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2–4 h and Brilliant Green 54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the halflife was found to be 60 h.     

Glycosides from <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

A new laboratory method for isolating the glycosides stevioside and rebaudiosides A and C from leaves of Stevia rebaudiana was proposed. According to HPLC, the glycoside contents in plants grown in Russia (Voronezh Oblast’) and Ukraine (Crimea) were 5–6% (stevioside) and 0.3–1.3% (rebaudiosides A and C). __________ Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 68–71, January–February, 2007     

Flavones from Callus Tissue of <Emphasis Type="Italic">Iris ensata</Emphasis>

Flavonoids uncharacteristic of intact plants were isolated from callus tissue of Iris ensata and were identified as 5-hydroxy-4′-methoxyflavone, 5-hydroxy-3′-methoxyflavone, and 5-hydroxy-2′-methoxyflavone using PMR and mass spectrometry. It was proposed that the lack of growth of callus tissue after changing cultivation conditions was related to the inhibiting effect of these flavones on cell proliferation. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 440–442, September–October, 2005.     

Detection of transferrin isoforms in human serum: comparison of UV and ICP–MS detection after CZE and HPLC separations

Two methods for separation of transferrin (Tf) sialoforms, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) with conventional UV absorbance detection, have been investigated and compared. First, conditions affecting the separation of the Tf isoforms by capillary zone electrophoresis and HPLC were carefully optimized. The use of 15 mmol L⁻¹ borate buffer (pH 8.4) containing 3 mmol L⁻¹ diaminobutane (DAB) as additive enabled good separation of the Tf isoforms by CE (75 cm×50 μm i.d. fused silica capillary) at 25 kV. In HPLC, a gradient of ammonium acetate (from 0 to 250 mmol L⁻¹ in 45 min) buffered at pH 6 (Tris-HCl) proved suitable for separation of Tf isoforms on a Mono-Q HR 5/5 anion-exchange column. On-line specific detection of the iron associated with the different Tf isoforms, after Fe saturation, by inductively coupled plasma mass spectrometry (ICP–MS) was studied in detail to compare its analytical performance with UV detection. For both CE and HPLC an octapole reaction system (ORS) ICP–MS instrument was used to minimize polyatomic interferences on the ⁵⁶Fe major isotope. Limits of detection of the different isoforms were in the range of 0.02–0.04 μmol L⁻¹ Tf for HPLC–ICP (ORS)–MS. This hybrid technique proved more selective and reliable detection of transferrin isoforms with 2, 3, 4, 5, and 6 sialic acid residues (S₂, S₃, S₄, S₅, and S₆) in real serum samples. Interesting results from iron speciation of Tf in serum from healthy individuals and from pregnant women are given.     

Biosynthesis of Cd-bound phytochelatins by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and their speciation by size-exclusion chromatography and ion-pair chromatography coupled to ICP–MS

Cd-bound phytochelatins (Cd–PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography–UV–Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma–mass spectrometry (ICP–MS) for analysis of Cd–PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd–PC signals. By use of isotopically enriched ¹¹⁶Cd–PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd–PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd–PCs are decisive factors in their detection.     

Production and partial characterization of extracellular peroxidases produced bystreptomyces avermitilis UAH30

The effect of a number of environmental parameters (pH, temperature, carbon and nitrogen ratio of nutrient) on the production of extracellular peroxidase enzymes byStreptomyces avermitilis UAH30 was examined. Maximum specific peroxidase activity (0.12 U/mg of protein) was obtained after 72 hours of 1 incubation at 45‡C in a minimal salt medium (pH 7.5) containing 0.6% (w/v) yeast extract and 0.6% (w/v) xylan corresponding to a C:N ratio of 4 to 1. A study of the effect of incubation on peroxidase activity showed that the enzyme was stable and active for at least one hour after incubation at 50‡C while at higher temperatures the stability and activity of the peroxidase was reduced such that at 60‡C the peroxidase activity has a half life of 20 min while at 80‡C the half life was reduced to 5 min. The activation energy for deactivation as a result of thermal denaturation of the enzyme was calculated to be 80 ±7 kJ/mol. The optimum pH for the activity occurred between a pH range of 6.5–8.5 with pKa₁ and pKa₂ of 5.1 ±0.1 and 9.7 ±0.1, respectively. The K_m and V_max for the peroxidase activity were determined to be 1.45 mM and 0.31 unit per mg protein respectively using 2,4dicholorophenol (2,4-DCP) as a substrate. Characterization of the peroxidase activity revealed activity against L,3–4 dihydroxyphenylalanine and guaiacol, while no inhibition of peroxidase activity could be detected with the haem inhibitors such as potassium cyanide and sodium azide, suggesting the lack of haem component in the tertiary structure. Aspects of using the crude peroxidase preparation in the pulp and paper industry are discussed.     

Chemical composition of <Emphasis Type="Italic">Capparis spinosa</Emphasis> fruit

The known compounds cappariloside A and stachydrin, an adenosine nucleoside, and for the first time from plants of the Capparidaceae family the known compounds hypoxanthine and uracil were isolated from Capparis spinosa (Capparidaceae) fruit. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 149–151, March–April, 2007.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Comparative analysis of essential oil compositions from leaves and stems of Rhododendron adamsii, R. aureum, and R. dauricum

Chemical compositions of essential oils (EO) from leaves and stems of Rhododendron adamsii, R. aureum, and R. dauricum were studied by GC/MS. The analysis demonstrated that the EO of these plants contained mainly mono-and diterpene hydrocarbons. A large amount of alkanes was also found in the EO of R. aureum. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 344–347, July–August, 2006.     

Multiresidue analysis of atrazine, diuron and their degradation products in sewage sludge by liquid chromatography tandem mass spectrometry

A multiresidue method has been developed to analyze atrazine (ATZ), diuron (DIU), and their major degradation products, desethylatrazine (DEA), desisopropylatrazine (DIA), and dichlorophenylmethylurea in sewage sludge. Liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI-MS–MS) allowed, in the multiple-reaction monitoring mode, the simultaneous analysis of these pesticides in only one run after their extraction with ethyl acetate–dichloromethane 90:10 (v/v) and a cleanup on a Florisil column. Stable isotopically labeled ATZ and DIU were used as internal standards to overcome matrix effects during the pesticide quantification. Using fortified samples, the method gave rise to 86–115% as mean recovery values depending on the analyte. Limits of detection (LODs) and of quantification (LOQs) ranging from 0.3 (DIA) to 1.5 (DEA) μg kg⁻¹ dw and from 0.4 (DIA) to 2.0 (DEA) μg kg⁻¹ dw, respectively, were sufficient to achieve the monitoring of these molecules in sludge from wastewater treatment plants of the Ile-de-France region.     

Laccase from Coriolus hirsutus as alternate label for enzyme immunoassay

A new label—laccase from the fungus Coriolus hirsutus—was applied for solid-phaseenzyme-linked immunosorbentassays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems perm it the detection of 2,4-D in concentrations down to 10–20ng/mL; time of the assays is 1.5–2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.     

Intensification of Biocatalytical Processes by Synergistic Substrate Conversion. Fungal Peroxidase Catalyzed <Emphasis Type="Italic">N</Emphasis>-Hydroxy Derivative Oxidation in Presence of 10-Propyl Sulfonic Acid Phenoxazine

Many industrial pollutants, xenobiotics, and industry-important compounds are known to be oxidized by peroxidases. It has been shown that highly efficient peroxidase substrates are able to enhance the oxidation of low reactive substrate by acting as mediators. To explore this effect, the oxidation of two N-hydroxy derivatives, i.e., N-hydroxy-N-phenyl-acetamide (HPA) and N-hydroxy-N-phenyl-carbamic acid methyl ester (HPCM) catalyzed by recombinant Coprinus cinereus (rCiP) peroxidase has been studied in presence of efficient substrate 3-(4a,10a-dihydro- phenoxazin-10-yl)-propane-1-sulfonic acid (PPSA) at pH 8.5. The bimolecular constant of PPSA cation radical reaction with HPA was estimated to be (2.5 ± 0.2)·10⁷ M⁻¹ s⁻¹ and for HPCM was even higher. The kinetic measurements show that rCiP-catalyzed oxidation of HPA and HPCM can increase up to 33,000 times and 5,500 times in the presence of equivalent concentration of high reactive substrate PPSA. The mathematical model of synergistic rCiP-catalyzed HPA–PPSA and HPCM–PPSA oxidation was proposed. Experimentally obtained rate constants were in good agreement with those calculated from the model confirming the synergistic scheme of the substrate oxidation. In order to explain the different reactivity of substrates, the docking of substrates in the active site of the enzyme was calculated. Molecular dynamic calculations show that the enzyme–substrate complexes are structurally stable. The high reactive PPSA exhibited higher affinity to enzyme active site than HPA and HPCM. Furthermore, the orientation of HPA and HPCM was not favorable for proton transfer to the distal histidine, and different substrate reactivity was explained by these diversities.     

Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Strategies for examination of Alzheimer’s disease amyloid precursor protein isoforms

We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC–MS, MALDI–MS, and MS–MS for isolation and identification of amyloid precursor protein (APP) isoforms APP₆₉₅, APP₇₅₁, and APP₇₇₀. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, a monoclonal antibody raised to amino acids 1–17 of the β-amyloid peptide sequence, which is present in all three alpha secretase-cleaved isoforms of interest. The next stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as “tags” for isoform detection.