I. Yu. Sakharov

Purification and substrate specificity of peroxidase from sweet potato tubers

Previously the screening of tropical plants demonstrated a high peroxidase activity in sweet potato (Ipomoea batatas) tubers. The major peroxidase pool is localized in peel. Using peel of sweet potato as a source, the sweet potato peroxidase (SPP) has been isolated and purified to homogeneity. The enzyme purification included homogenization, extraction of colored compounds and consecutive chromatographies on Phenyl-Sepharose and DEAE-Toyopearl. The purified SPP had specific activity of 4900 U mg(-1) protein, RZ (ratio of absorbances at 403 and 280 nm, respectively) 3.4, molecular mass of 37 kDa and isoelectric point of 3.5. The spectrum of peroxidase from sweet potato is typical for plant peroxidases with a Soret maximum at 401 nm and the maxima in the visible region at 497 and 638 nm, respectively. The substrate specificity of SPP is distinct from the specificity of other plant peroxidases, ferulic acid being the best substrate for SPP. (Less)

Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was ∼40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.

Palm Tree Peroxidases

Over the years novel plant peroxidases have been isolated from palm trees leaves. Some molecular and catalytic properties of palm peroxidases have been studied. The substrate specificity of palm peroxidases is distinct from the specificity of other plant peroxidases. Palm peroxidases show extremely high stability under acidic and alkaline conditions and high thermal stability. Moreover, these enzymes are more stable with respect to hydrogen peroxide treatment than other peroxidases. Due to their extremely high stability, palm peroxidases have been used successfully in the development of new bioanalytical tests, the construction of improved biosensors, and in polymer synthesis.

Long-Term Chemiluminescent Signal Is Produced in the Course of Luminol Peroxidation Catalyzed by Peroxidase Isolated from Leaves of African Oil Palm Tree

Optimal conditions were found for the oxidation of luminol by hydrogen peroxide in the presence of peroxidase isolated from leaves of the African oil palm tree Elaeis guineensis (AOPTP). The pH range for maximal chemiluminescence intensity (8.3-8.6) is similar for AOPTP, horseradish, and Arthromyces ramosus peroxidases and slightly different from that for tobacco peroxidase (9.3). Increasing the buffer concentration decreases the chemiluminescence intensity. As in the case of other anionic peroxidases, the catalytic efficiency of AOPTP does not depend on the presence of enhancers (4-iodophenol and 4-hydroxycinnamic acid) in the reaction medium. The detectable limit of AOPTP assayed by luminol peroxidation is 2·10–12 M. The long-term chemiluminescence signal produced during AOPTP-dependent luminol peroxidation is a characteristic feature of the African oil palm enzyme. This feature in combination with its very high stability suggests that AOPTP will be a promising tool in analytical practice.

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.

Enzymatic synthesis of a conducting complex of polyaniline and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) using palm tree peroxidase and its properties

An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.

Stabilization of proteins by modification with water-soluble polysaccharides

Abstract The effect of chemical modification by water-soluble polysaccharides on the thermostability of basic pancreatic trypsin (EC 3.4.21.4) inhibitor (BPTI) has been studied. Stabilization of BPTI was achieved by multipoint protein-matrix interaction with the formation of new linkages. The nature of the formed bonds is covalent after modification by bi- and/or polyfunctional reagents and ionic after preparation of polyelectrolytic complexes between BPTI and soluble polysaccharides. The thermostability of BPTI increased because of protein-protein interaction on the water-soluble carrier. This approach may be generally employed for the preparation of stabilized water-soluble proteins.

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.

Phenothiazine derivatives as enhancers of peroxidase-dependent chemiluminescence

Some N-alkyl phenothiazines with different ionic groups were studied as enhancers of chemiluminescence catalyzed by soybean peroxidase. It was shown that under experimental conditions, the compounds with positively charged groups do not exhibit enhancing ability, while the addition of phenothiazines with negatively charged groups to a substrate mixture significantly increased the chemiluminescence intensity. The relationship between the enhancing activity of phenothiazines and their capacity for enzymatic oxidation by hydrogen peroxide was found. The enhancers discovered new opportunities for increasing the sensitivity of determination of analytes by chemiluminescent enzyme immunoassay.

Enzyme immunoassay for the determination of hexestrol in meat

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an anabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20–IC80) equaled 0.03–0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.26–32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.