Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry

Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes.     

Some characteristics of peroxidase secreted by cotton ovule cultures

Cotton (Gossypium hirsutum L.) ovule cultures secreted a soluble peroxidase into the surrounding medium, resulting in a 200-fold increase in this activity during the 30-day growth period. The peroxidase activity was thermostable from 4°C to 60°C and displayed a pH optimum of 5.5 to 6.0. The ovule peroxidase was susceptible to periodate treatment and very resistant to protease digestion. The data suggest that the peroxidase activity is a glycoprotein. Interpretation of peroxidase data may be complicated by the presence of phenol oxidase activity in the same preparations. The presence of phenol oxidases was ruled out by the inaction of a tyrosinase-specific inhibitor, tropolone.Abbreviations PMSF phenylmethylsulfonyl fluoride     

Changes in the activities of catalase, peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Observations on the Germ Aleurone of Barley. Phenol Oxidase and Peroxidase Activity

Strips of tissue containing the germ aleurone layer were removedfrom dry, harvest-ripe grains of barley (Hordeum vulgare L.)and incubated in buffered solutions of phenolic compounds, withand without the addition of hydrogen peroxide. Peroxidase ando-diphenol oxidase activity were found in the material releasedinto the incubation medium, and in the cytoplasm of the germaleurone cells. Peroxidase activity was located in the cellwalls and appeared to be high in the region where the germ aleuronecovering the embryonic axis merges into that which adheres tothe scutellum i.e. the region in which a row of germ aleuronecells becomes lignified following germination. Monophenol oxidaseactivity was not detected in the released enzymes or in theintact tissue. Although hydroquinone was oxidized in the cytoplasmof the germ aleurone tissue, unequivocal evidence of the presenceof laccase was not obtained. The oxidation of endogenous phenolicsubstances by phenol oxidases and peroxidases is discussed inrelation to anti-microbial defence mechanisms which appear tooperate in the germ aleurone during germination.Copyright 1994,1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, catechol oxidase, laccase, peroxidase, defence mechanisms, germination     

The effect of carbamide and thiocarbamide on the resistance of apple to apple scab

Carbamide and thiocarbamide decreased the resistance of apple to apple scab when infiltrated into apple leaves prior to infection with the disease. In three apple varieties these two substances strikingly stimulated infection with two monosporic isolates of the fungusVenturia inaequalis (Cke.) Wint. It was established that both carbamide and thiocarbamide inhibited polyphenol oxidase activity in apple leavesin vivo, butin vitro only thiocarbamide was inhibitory. It can be concluded that the effect on apple resistance to apple scab is based on an inhibition of polyphenol oxidase.     

A new phenol oxidase produced during melanogenesis and encystment stage in the nitrogen-fixing soil bacterium Azotobacter chroococcum

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.     

Soluble and immobilized phenol oxidase of the fungusMycelia sterilia IBR 35219/2: A comparative study

Phenol oxidase (EC 1.14.18.1) from the microscopic fungusMycelia sterilia IBR 35219/2 was immobilized using glutaraldehyde on macroporous silica carriers. The enzyme immobilized on amino-Silochrome SKh-2 or aminopropyl-Silochrome 350/80 exhibited maximum activity. Soluble and immobilized phenol oxidases were compared. Compared to the soluble enzyme, the activity of which was optimum at pH 5.5, immobilized phenol oxidase exhibited optimum activity under slightly more acidic conditions (pH 5.2). Immobilization considerably increased enzyme stability. Both soluble and immobilized forms of phenol oxidase fromM. sterilia IBR 35 219/2 catalyze oxidative conversion of phenolic compounds of green tea extract.     

Induction of defence response against blister blight by calcium chloride in tea

Tea, the second most consumed beverage after water in the world, is produced from the processed tender leaves of tea plants (Camellia sinensis). Production of tea is hindered by various biotic and abiotic stresses. Among the biotic factors, blister blight disease caused by an obligate fungal pathogen, Exobasidium vexans Massee, is a serious problem to the tea industry. The present study was to evaluate the efficacy of elicitor calcium chloride (CaCl₂) in inducing resistance in tea plants against blister blight disease. During the pick time of blister incidence at Darjeeling tea garden, the application of CaCl₂ at a concentration of 1% resulted in disease inhibition around 71% over the control set. Treatment also resulted in the induction of defence enzymes like peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-1,3-glucanase and higher phenol accumulation. Furthermore, the increase in defence molecules also correlated with increase in nitric oxide (NO) generation, a potent defence molecule in plant defence. The result suggests that CaCl₂ can used as a potential elicitor in the integrated disease management in organic tea cultivation.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Peroxidases and lignification in relation to the intensity of water-deficit stress in white clover (Trifolium repens L.)

To investigate the lignification process and its physiologicalsignificance under drought-stressed conditions, the changesin enzymes responsible for lignification and the related physiologicalparameters were determined in white clover (Trifolium repensL.) leaves during 28 d of water deficit treatment. Water deficitgradually decreased leaf water potential (_w) to –2.33MPa at day 28. For the first 14 d of water deficit, ascorbateperoxidase and phenylalanine ammonia-lyase were highly activated.Neither a change in the parameters symptomatic of oxidativestress nor growth inhibition was observed. The reduction ofleaf biomass occurred from 21 d of water deficit treatment when_w was –2.27 MPa or less, and was concomitant with theincrease of lipid peroxidation and lignin content. As _w decreasedbelow –1.67 MPa from 14 d of water deficit, the enhancedactivation of guaiacol peroxidase, coniferyl alcohol peroxidase,syringaldazine peroxidase, and benzidine peroxidase was involvedin lignification rather than in protection of plant tissuesagainst the oxidative damage. The data indicate that a highactivation of lignifying enzymes during terminal stress maybe a drought stress-induced injurious symptom, which leads toreduced forage growth and digestibility. Key words: Drought, lignification, peroxidases, phenylalanine ammonia-lyase, polyphenol oxidase, Trifolium repens Received 23 July 2006; Revised 18 November 2006 Accepted 20 November 2006     

Creation of Bifunctional Indicating Complex Based on Nanodiamonds and Extracellular Oxidases of Luminous Fungus <Emphasis Type="Italic">Neonothopanus nambi</Emphasis>

A bifunctional indicating complex was created by immobilization of extracellular oxidases (glucose oxidase and peroxidases) of luminous fungus Neonothopanus nambi onto modified nanodiamonds (MNDs) synthesized by detonation. It was found that the enzymes firmly adsorb onto MND particles and exhibit their catalytic activity. Model in vitro experiments showed that the created MND–enzymes complex is suitable for repeated use for analyte (glucose and phenol) testing and retains its activity after storage at 4°C in deionized water for 1 month. The data obtained offer the prospects for developing a new class of reusable multifunctional indicating and diagnostic test systems on the basis of MNDs and higher fungal enzymes for medical and ecological analytics.     

Enhancement of Peroxidase, Polyphenol Oxidase and Superoxide Dismutase Activities by Triadimefon in NaCl Stressed Raphanus Sativus L.

The activities of peroxidase, polyphenol oxidase and superoxide dismutase was significantly lower in roots and leaves of NaCl stressed radish (Raphanus sativus L.) plants. Addition of triadimefon to the NaCl stressed plants increased peroxidase, polyphenol oxidase and superoxide dismutase activities, and thereby ameliorated the negative effect of NaCl stress.     

Peroxisomal enzyme activities in attached senescing leaves

Recently it has been demonstrated that detached leaves show glyoxysomal enzyme activities when incubated in darkness for several days. In this report glyoxylate-cycle enzymes have been detected in leaves of rice (Oryza sativa L.) and wheat (Triticum durum L.) from either naturally senescing or dark-treated plants. Isolated peroxisomes of rice and wheat show isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2) and -oxidation activities. Leaf peroxisomes from dark-induced senescing leaves show glyoxylic-acid-cycle enzyme activities two to four times higher than naturally senescing leaves. The glyoxysomal activities detected in leaf peroxisomes during natural foliar senescence may represent a reverse transition of the peroxisomes into glyoxysomes.This work was supported by CNR Italy, special grant RAISA, subproject 2, paper no. 26.     

Induced resistance against Alternaria brassicae blight of mustard through plant extracts

An experiment was conducted to study the effect of plant extracts on soluble sugar, soluble phenol and defence-related enzymes response against Alternaria blight in mustard crop. The efficacy of six selected plant extracts (5 and 10%) used as foliar sprays at 60 and 70 days after sowing and mustard leaves was used for investigation. The results indicate that soluble phenol and sugar content in mustard leaves significantly increases in response to spraying of Azadirachta indica seed kernel, Calotropis procera and A. indica leaf extracts. The soluble protein, viz. peroxidase, polyphenol oxidase and phenylalanine ammonia lyase content, was higher in mustard leaves sprayed with C. procera leaves extract, A. indica seed kernel and Allium sativum bulb extract.     

Changes in the Activity of Antioxidant Enzymes in Wheat Leaves and Roots as a Function of Nitrogen Source and Supply

The activity of enzymes participating in the systems of antioxidant protection was assayed in the second leaf and roots of 21-day-old wheat seedlings (Triticum aestivum L.) grown in a medium with nitrate (NO^– ₃ treatment), ammonium (NH⁺ ₄ treatment), or without nitrogen added (N-deficiency treatment). The activities of superoxide dismutase (SOD), peroxidase, ascorbate peroxidase, glutathione reductase, and catalase in the leaves and roots of the NH⁺ ₄ plants was significantly higher than in the plants grown in the nitrate medium. The activity of SOD decreased and ascorbate peroxidase markedly increased in leaves, whereas the activity of ascorbate peroxidase increased in the roots of N-deficient plants, as compared to the plants grown in nitrate and ammonium. Low-temperature incubation (5°, 12 h) differentially affected the antioxidant activity of the studied plants. Whereas leaf enzyme activities did not change in the NH⁺ ₄ plants, the activities of SOD, peroxidase, ascorbate peroxidase, and catalase markedly increased in the NO^– ₃ plants. In leaves of the N-deficient plant, the activity of SOD decreased; however, the activity of other enzymes increased. In response to temperature decrease, catalase activity increased in the roots of NO^– ₃ and NH⁺ ₄-plants, whereas in the N-deficient plants, the activity of peroxidase increased. Thus, in wheat, both nitrogen form and nitrogen deficiency changed the time-course of antioxidant enzyme activities in response to low temperature.     

Extracellular Oxidases of the Lignin-Degrading Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis>

Two extracellular oxidases (laccases) were isolated from the extracellular fluid of the fungus Panus (Lentinus) tigrinus cultivated in low-nitrogen medium supplemented with birch sawdust. The enzymes were purified by successive chromatography on columns with TEAE-cellulose and DEAE-Toyopearl 650M. Both oxidases catalyze oxidation of pyrocatechol and ABTS. Moreover, oxidase 1 also catalyzes oxidation of guaiacol, o-phenylenediamine, and syringaldazine. The enzymes have identical pH (7.0) and temperature (60–65°C) optimums. Absorption spectra of the oxidases differ from the spectra of typical “blue” laccases and are similar to the spectrum of yellow oxidase.__________Translated from Biokhimiya, Vol. 70, No. 6, 2005, pp. 850–854.Original Russian Text Copyright © 2005 by Cadimaliev, Revin, Atykyan, Samuilov.     

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons

Crude particulate homogenates from leaves of barley (Hordeum vulgare L.), rice (Oryza sativa L.), leaf-beet (Beta vulgaris var.cicla L.) and pumpkin (Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm^–3 and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta176, 548–550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence).We are grateful to Professor P. Matile (Zürich, Switzerland) for his encouragement in pursuing this work.     

Oxidative enzymes activities of mycorrhizal inoculated pepper plant infected with phytophthora infestans

The activities of polyphenol oxidase, peroxidase and catalase were investigated in Glomus etunicatum an arbuscular mycorrhizal inoculated pepper plant infected with Phytophthora infestans. Pepper plant inoculated with pathogen alone, simultaneously inoculated with mycorrhizal and pathogen or dually inoculated with pathogen before mycorrhizal had increased level of the oxidative enzymes activities especially at 4 weeks after planting. Of the three oxidases investigated catalase had the highest level of activities in all the treatments while the level of peroxidase was the lowest. The results showed that pathogen alone, build up the level of the oxidases in the inoculated pepper plant while inoculation of the pepper plant with AM mycorrhizal either simultaneously or dually with pathogen lower the activity of the oxidases indicating a passive protective effect of mycorrhizal.     

Enzymes of the xylotrophic basidiomycete Lentinus edodes F-249 in the course of morphogenesis

It has been shown that the fungus Lentinus edodes grown on a solid wort agar substrate produces intracellular enzymes, including Mn-dependent peroxidase, laccase, and tyrosinase as a family of isoforms. The composition of the complex (containing one to four forms of each enzyme) varied during the basidiomycete life cycle. The activity of oxidases was maximal at the stage of nonpigmented mycelium and at the stages of a brown mycelial mat and a fruit body. The activity of tyrosinase increased in the course of mycelium pigmentation and had two maxima: at the stage of a brown mycelial mat and at the stage of a fruit body. Laccase and tyrosinase activities were shown to increase sharply upon addition of oak sawdust extract to the culture medium as compared with the enzyme activities of mycelium grown on wort agar alone. It was established that the effect of phenol oxidase substrates on the growing mycelium consists in a twofold acceleration of the process of morphogenesis in the fungus L. edodes.     

On some biochemical physiognomies of two common Darjeeling tea cultivars in relation to blister blight disease

Abstract

Tea, Camellia sinensis (L.) O. Kuntze, an agro-based industry, is considered as one of the prime sector of exporting resources and thus considered as “cash-crop”. Earlier report shows that tea is native to eastern and northern India, which was cultivated and consumed there since long back. Presently, more scientific reports confirmed the health-benefit traits of tea and awareness increased to a greater extent and in this regard, tea has gained its best worldwide popularity. Darjeeling Tea attained its highest acceptance globally for its pre-eminence in flavour, colour and taste and thus crop improvement is the prime interest to the Indian Scientific Community. Blister blight disease, a common disorder of tea bushes (Exobasidium vexans, a Basidiomycetes fungus) causes drastic damage of tea plantation. Depending on quality production, two common cultivars were released by TRA, Jorhat, Assam viz. Bannockburn – 157 (B-157) and Ambei Valai - 2 (AV-2), of which B-157 is susceptible to the Blister Blight and AV-2 is fairly resistant cultivar. Some biochemical considerations between the two cultivars have been made here for understanding the biochemical reasons behind the resistant characteristics. Plant secondary metabolites, like total phenol, proanthocynadin, total soluble protein provide defending feature against disease onset. AV-2 cultivar shows advantage over B-157 in these regard. Depending on band intensity analysis of native gels, acid phosphatase, catechol oxidase, peroxidase and superoxide dismutase occur in superior amount in AV-2 cultivar than that of B-157. The specific role of these enzymes in blister blight disease compatibility of two cultivars studied has been discussed.