Search

Trametes versicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase Tv DyP1 and the versatile peroxidase Tv VP2. Both peroxidases were heterologously produced in Escherichia coli , biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and Tv DyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while Tv VP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and Tv DyP1 was globally found to be more stable than Tv VP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of Tv VP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, Tv DyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and Tv VP2 was shown to accept a wider range of reducing substrates than Tv DyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with Tv VP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and Tv VP2 presented a greater ability to oxidize and decolorize the dye substrates than Tv DyP1. IMPORTANCE Trametes versicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor , one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.

Contact: nawel.achir@supagro.inra.fr. Fax: 133-467-617055; International audience; The kinetics of trans-beta-carotene and trans-lutein degradation were individually investigated in palm olein and Vegetaline (R), at four temperatures ranging from 120 to 180 degrees C. HPLC-DAD analysis was carried out to monitor trans and cis carotenoid variations over the heating time at each temperature. In both oils, initial trans-beta-carotene and trans-lutein degradation rates increased with temperature. Trans-lutein was found to degrade at a slower rate than trans-beta-carotene, suggesting a higher thermal resistance. The isomers identified were 13-cis- and 9-cis-beta-carotene, and 13-cis-, 9-cis-, 13'-cis-, and 9'-cis-lutein. In spite of the higher number of lutein cis isomers, their total amount was lower than that of beta-carotene cis isomers. Trans and cis carotenoids were involved in degradation reactions at rates that increased with temperature. All degradation rates were generally found to be lower in Vegetaline (R) than in palm olein. These results were explained by the initial composition of the two oils and especially their peroxide and vitamin E contents.

At the industrial scale, the chemical Prileshajev reaction is currently the only method applied to produce epoxidized plant oils. Using enzymes could be an alternative way allowing an environmentally benign and more selective epoxidation reaction. P450 monooxygenases, diiron-center oxygenases, lipoxygenases, peroxygenases, and hydrolases performing perhydrolysis are enzyme classes involved in free fatty acid and glyceride epoxidation. After a brief description of these biocatalysts, this review focuses on the chemo-enzymatic epoxidation of unsaturated fatty acyd chains, where a lipase-catalyzed peroxy acid formation is followed by an uncatalyzed “self-epoxidation”. The molecular bases of lipase-catalyzed perhydrolysis as well as the different parameters influencing the epoxidation reaction are reviewed and described in details.

The antibacterial activity of the lactoperoxidase system (LPS) on the growth of Xanthomonas campestris, the causal agent of bacterial black spot in mangoes, Botryodiplodia theobromae, the causal agent of stem-end rot disease in mangoes, and Colletotrichum gloeosporioides, the causal agent of anthracnose disease in mangoes, was determined during culture at 30 degrees C and at several pH values (4.5, 5.5, and 6.5). When the results of using the LPS were compared with those from control cultures without the LPS reagents, the growth of the three microorganisms was totally inhibited in all of the conditions tested. Viability tests enumerating cultivable cells of X. campestris showed that the LPS had a bactericidal effect, whatever the pH value. This effect is faster at pH 5.5, corroborating the results reported in the literature (optimal pH for the LPS efficiency). Further, we proved that hydrogen peroxide alone had little inhibition effect on the growth of the microorganisms studied. This compound is essentially used to convert thiocyanate into hypothiocyanate during the lactoperoxidase reaction. The potential of the LPS for the postharvest treatment of the fruits for controlling microbial diseases was thus demonstrated. Nevertheless, further studies are needed on fresh fruits before envisaging any application.

The overall objective of this research was to find a new way to valorize rye bran, by producing a gellifier from the enzymatic solubilization of arabinoxylans (AX). The effects of three pure endo-xylanases from Aspergillus niger (Xyl-1), Talaromyces emersonii (Xyl-2), and Bacillus subtilis (Xyl-3) and of Grindamyl S100 (GS100), a commercial enzyme preparation containing a Xyl-1 type endo-xylanase, were tested on rye bran to study the solubilization of water-unextractable arabinoxylans (WUAX). Eight different extrusion-treated rye brans were also used as substrates to find the best physical treatment to facilitate enzymatic arabinoxylan (AX) solubilization. Arabinoxylans were better solubilized from the bran extruded at high temperature using Xyl-3. This enzyme was then tested in combination with pure (1,4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and endo-beta-d-glucanase or ferulic acid esterase (FAE), from A. niger. Only beta-glucanase in combination with Xyl-3 improved the AX extraction, but it did not have a marked effect on the viscosity of the extracts. Xyl-3 was then tested on a high-shear-treated rye bran, and results were compared to those obtained with the high-temperature-extruded rye bran. The high-shear treatment did not improve the bran AX enzymatic solubilization. The combination of FAE with Xyl-1 or Xyl-3 did not improve the AX extraction from untreated and high-shear-treated rye bran. Finally, to study the gelation capacity of the enzymatically solubilized AX, the effect of the hydrogen peroxide/horseradish peroxidase (H(2)O(2)/POD) was tested on the Xyl-3 high-temperature-extruded bran extracts. Solubilized AX did not gel in the presence of the oxidizing system.

Colour and texture are important features of amala, a traditional thick paste obtained from dry yam flour. Tubers from eight yam cultivars were blanched at 65 °C for 20 min and dried at 40 °C for 5 days to test biochemical, thermal and pasting changes occurring during traditional yam flour processing. Blanching reduced peroxidase activity and drying reduced polyphenoloxidase activity, but total phenol content and the brown index of flour and of amala increased dramatically during the latter operations. The brown index of amala was significantly correlated with the total phenol content of the flour (r=0.84) and the peroxidase activity of the fresh tubers (r=0.75). Amylose content and starch gelatinization enthalpy remained stable. For all cultivars, drying significantly increased the onset gelatinization temperature, suggesting the occurrence of starch annealing. The latter leads to a reduction in swelling power and solubility during pasting, and hence to a lower paste viscosity.

9 páginas, 8 figuras, 2 tablas -- PAGS nros. 303-311, The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn2+. Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds, This work has been carried out with funding from the EU FP7 project “Peroxicats” (KBBE-2010-4-265397), the Spanish Ministry of Science and Innovation (BIO2009-08446, PRI-PIBAR-2011-1402), and the project “Lignin Fibre” financed by the Academy of Finland (Grant number 133091)

Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of the phenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux through the pathway is not that well established. The aim of this study was to use a strawberry (Fragaria × ananassa) microarray to investigate gene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit. An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken to identify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed 15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results were validated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than 900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberry fruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait. Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux from anthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The list of the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used to develop molecular markers to select phenolics-rich germplasm., This work was supported by PLANT-KBBE FraGenomics, by the Euroinvestigacion Subprogram from PLANT_KBBE Call, provided by the Spanish Science and Technology Ministry (grant no. EUI2008–03770 to A.M.), by the Centre CONSOLIDER on Agrigenomics and the Xarxa de Referencia en Biotecnología of the Generalitat de Catalunya, and by the Deutsche Forschungsgemeinschaft (grant no. SCHW634/14 to S.-Y.Y. and W.S.).

Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β- D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous β-glucosidase, liberates vanillin, the major aroma component of vanilla. Little is known on the spatial distribution of aroma-generating phenolics, and the enzymes responsible for their liberation (β-glucosidase) and oxidation (peroxidase). We report here quantitative data with respect to these three components in relation to the anatomy of the pod. Furthermore, the spatial progression of oxidation is shown. Materials and methods. Mature green vanilla pods were analyzed for their contents of phenolics (HPLC), and β-glucosidase and per- oxidase activities by spectrophotometric techniques using p-nitrophenyl glucoside and vanillin as sub- strates, respectively. Lipids were examined under fluorescence microscopy after Nile red staining. Oxidation development was observed on transverse slices of pods. Results and discussion. Phenolics, and β-glucosidase and peroxidase activities showed gradients of increasing-decreasing concentrations from the stem to the blossom end of pods. The β-glucosidase activity is distributed in between the pla- centae, mesocarp, and trichomes in a ( 7/2/1 ) proportion while that of peroxidase shows a (38 / 1) ratio inthe mesocarp and placentae, and was absent from trichomes. Oxidation begins from the blossom end in the placentae, progressively invading the mesocarp and moving towards the stem end. Con- clusion. The green mature vanilla pod is spatially heterogeneous for its phenolics, and β-glucosidase and peroxidase activities, its placentae playing an important role in the liberation of vanillin and its sub- sequent oxidation. France / Vanilla planifolia / Orchidaceae / vanilla / β-glucosidase / peroxidase / glucovanillin / vanillin / lipids / oxidation / gradient / mesocarp / placentae / trichomes / xylem

Bayoud, caused by #Fusarium oxysporum# f. sp.# albedinis# (Foa), is the most damaging disease of date palm in Morocco. In the present study we have investigated the effect of jasmonic acid (JA) on two defencerelated enzymes, namely peroxidases (POX) and polyphenoloxidases (PPO) in date palm seedlings root. Our data show that exogenous application of JA at a concentration of 50 [mu]M increased the activity of both enzymes. The increase of POX activity in the presence of JAwas much more important than that observed following infection with the pathogen. As compared to untreated plants, PPO activity was 2.2 and 1.3 times higher in BSTN and JHL cultivars respectively. In addition, PAGE analysis revealed increased band intensity of the major constitutive isoforms of POX and PPO in both JA-treated and Foa-treated seedlings. Close examination of symptomatic and asymptomatic plants showed that root tissues of symptomatic plants were massively colonized by Foa. Also, disease development in these plants appeared to involve a marked degradation of the host cell walls early during the process of pathogen invasion. In contrast, the presence of Foa in asymptomatic plants induced limited necrotic lesions (hypersensitive-reaction like lesions) that were probably involved in reducing the progression of the pathogen. Together, our findings indicate that JA is capable of enhancing date palm root resistance to infection by Foa via the activation of defence-related enzymes such as PPO and POX.

A postharvest treatment based on citric acid and chitosan was tested on two litchi cultivars of different provenance. Its effects on the activities of polyphenol oxidase (PPO), peroxidase (POD) and anthocyanase and on the anthocyanin content of the pericarp were measured as factors responsible for pericarp browning. The red colour of the pericarp was measured during storage of the fruit. The major anthocyanins present in both cultivars were cyanidin-3-rutinoside and cyanidin-3-glucoside. Although the concentration of cyanidin-3-rutinoside was 64% lower in the cultivar Kwai may (Guiwei) than in Wai chee (Huaizhi), this component represented more than 90% of total anthocyanins in both cultivars. The activity of PPO was six times greater in Kwai may than in Wai chee and the activity of POD was 30-times greater. The activity of POD was greater than that of PPO in both cultivars. The two cultivars, which differ in anthocyanin and oxidative enzyme compositions, responded differently to the acid and chitosan treatment, with the result that the red colour of Kwai may was better preserved during storage than that of Wai chee. This technique could be a future replacement for current sulphur treatments used to treat litchis transported by sea.

This study was undertaken to follow the kinetics of polyphenoloxidase (PPO), peroxidase (POD), and phenolic compounds during yam blanching at different temperatures and after drying and to identify by high-performance liquid chromatography (HPLC) the main phenolic compounds present in yam products. PPO activity was 50% higher in nonprocessed freeze-dried Florido (Dioscorea alata) than in nonprocessed freeze-dried Deba (Dioscorea rotundata). It decreased progressively during blanching. Forty-five percent of PPO activity remained after 50 min of blanching at 60 or 65 degrees C, whereas the POD activity dropped sharply to less than 20% of the initial activity after 10 min of blanching, whatever the blanching temperature. No anthocyanidins could be detected by HPLC at 520 nm in nonprocessed freeze-dried yam. Flavanols with a maximum absorption wavelength (lambda(max)) at 280 nm and cinnamic acid compounds with 320 nm lambda(max) were detected. Catechin was identified as the major flavanol with concentrations ranging from 0.26 to 0.41 microM g(-)(1) depending on cultivar. One cinnamic compound, ferulic acid, was identified and assessed in both cultivars (0.03-0.04 microM g(-)(1)). Total phenol, flavanol, and cinnamic contents decreased during blanching independently of temperature whereas some unresolved peaks were detected by HPLC in dried product. The latter was probably due to the consumption of coloring precursors and the appearance of polymerized or complex colored products.

In West Africa, yams are often processed into dry flour that is consumed as a thick paste (amala), the main quality attributes of which are colour, texture and taste. The traditional process consists of peeling, blanching and drying the fresh tubers. The fresh tubers may also be stored for several months before processing. This study was undertaken to test whether fresh tuber storage and pre- and post-blanching treatments (1-h storage of sliced fresh yam pieces in air or water and steeping in the blanching water, respectively) affect the physicochemical characteristics and pasting behaviour of dry yam flour produced from D. rotundata cultivars. Both pre-blanching treatments significantly reduced only peroxidase activity, while the post-blanching treatment significantly reduced polyphenoloxidase activity. Despite the effects on enzymatic activities, neither pre- nor post-blanching treatments had any significant effect on the amala brown index or the flour total phenol content. However, peak areas of three major cinnamic acid compounds, detected by HPLC, decreased continuously with steeping time after blanching, while fresh tuber storage led to a dramatic reduction in amala paste viscosity.

Following induction by auxin treatment, the rooting performance oi #Elaeis guineensis# Jacq. shoots obtained through #in vitro# clonal propagation by somatic embryogenesis varies considerably. This study involved a preliminary evaluation of guaiacol-peroxidase activity as a marker of #in vitro# rooting. It was carried out on 17 different clones obtained through a large-scale micropropagation procedure. No rooting occurred in the absence of exogenous auxins. Peroxidase activity evolved as previously described on other species, peaking between 10 and 14 days under optimum initiation conditions. The peak was found to shift in time when the rooting success rate was low. Peroxidase activity measurements, carried out prior to induction on 24 production batches belonging to 13 different atonal lines, revealed a significantly greater heterogeneity in batches with a low rooting success rate (< 50%). Several improved protocols aimed at optimising the rooting phase under large scale propagation conditions are proposed.

On estates, the rubber-tree, Hevea brasiliensis , is subject to a variety of diseases. Among them, root rots caused by Rigidoporus lignosus and Phellinus noxius are responsible for severe losses. When these parasites infect the roots they induce an increase in the peroxidase activity in the host tissues. Similar increases are shown in reaction zone tissues developed in response to Sphaerostilbe repens injury and in similar zones sometimes developed in R. lignosus infection, but then always devoid of any parasite. Comparative biochemical studies of the peroxidases from different types of tissue, including chromatographic separation into different peroxidase fractions, purification of one of the isoperoxidases and determination of some of its physico-chemical characteristics, lead to the following conclusions: (a) one isoperoxidase is mainly responsible for the increase in enzyme activity; (b) this isoenzyme is synthesized, probably de nova , by the host, not by the parasites, and thus represents a host reaction to parasite infection; (c) the presence of the parasite causes the tissue to produce this particular isoenzyme in excess of the amount normally found in healthy tissue; (d) this isoperoxidase polymerizes coniferyl alcohol, therefore it may contribute to the lignification reaction which was previously shown to occur. Thus, the peroxidase reaction may be involved, among others, in the host defence mechanisms.

17 páginas.-- 13 figuras.-- 104 referencias.-- . Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.biotechadv.2017.06.003, Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations., This work has been funded by the INDOX European project (KBBE-2013-7-613549), together with the BIO2014-56388-R and AGL2014-53730-R projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the BBI JU project EnzOx2 (H2020-BBI-PPP-2015-2-720297). The work conducted by the US DOE JGI was supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231.