Obtenção de nova fonte de peroxidase de folha de Copaifera langsdorffii Desf. com alta atividade

Objetivou-se neste trabalho extrair peroxidase de folha de Copaifera langsdorffii (COP), medir sua atividade, compará-la com a peroxidase de raiz forte (Horseradish peroxidase - HRP) e determinar o pH ótimo, a melhor solução extratora e o efeito de aditivos sobre a atividade da COP. Os resultados mostraram que a COP atingiu 81,6% da atividade de HRP e a faixa de pH ótimo foi de 5,5 a 6,0. A melhor solução extratora da enzima foi o tampão fosfato de sódio 50 mM, pH 6,0 e o melhor aditivo foi o PVPP. Concluindo, a COP apresenta atividade mais alta que outras peroxidases de diferentes fontes citadas na literatura.

Extração e caracterização parcial de peroxidase de folhas de Copaifera langsdorffii Desf.

Em recentes publicações têm sido descritos vários processos para obtenção de peroxidases. O propósito deste trabalho foi extrair peroxidase de folhas de Copaifera langsdorffii e caracterizar parcialmente a enzima usando planejamento experimental e teste univariado, para confirmação dos resultados obtidos por planejamento experimental. A atividade da peroxidase foi medida usando sistema guaiacol: peróxido de hidrogênio. A peroxidase isolada apresentou 81,6% da atividade da horseradish peroxidase e é de fácil obtenção, a partir de folhas de uma árvore abundante em todo o país. A peroxidase semi-purificada (COP) foi obtida pela precipitação do extrato bruto com acetona 65% (v.v-1), produzindo o pó cetônico. A COP apresentou atividade ótima na faixa de pH 5,0 a 7,0 e temperatura de 5 a 45 °C, com atividade máxima em pH 6,0 e 35 °C. A enzima mostrou-se estável em temperaturas inferiores a 50 °C e pH entre 4,5 e 9,0, por até 24 horas. A peroxidase foi inativada após 4 horas a 80 °C e após 3 minutos a 96 °C. Esta enzima demonstra possibilidade para ser usada como reagente para diagnósticos, construção de biossensores e outros métodos analíticos em vários campos da ciência.

Atividades das enzimas peroxidase (POD) e polifenoloxidase (PPO) nas uvas das cultivares benitaka e rubi e em seus sucos e geléias

A peroxidase e a polifenoloxidase estão relacionadas com o escurecimento de frutas, por isso o controle das atividades destas enzimas é de grande importância para a tecnologia de alimentos. Neste trabalho estudaram-se as atividades dessas enzimas nas uvas frescas das cultivares Benitaka e Rubi, bem como as suas termoestabilidades e as suas atividades enzimáticas residuais no suco e nas geléias (extra e light). Foi também avaliada a qualidade microbiológica dos produtos elaborados. As atividades da enzima POD, tanto da fração solúvel quanto da ionicamente ligada, foram semelhantes nas uvas das duas variedades, Benitaka e Rubi. A atividade da enzima polifenoloxidase foi maior na variedade Rubi. As operações de cocção e pasteurização foram mais eficientes para baixar as atividades enzimáticas residuais da POD e PPO quando aplicadas às geléias de uva, em comparação com o suco. Embora não foram suficientes para a total inativação enzimática, essas operações reduziram-nas consideravelmente, e foram eficientes para garantir seguridade microbiológica dos produtos, geléias e suco.

Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill.)

The aim of the present investigation was to evaluate the enzymatic activity of polyphenoloxidase and peroxidase in avocado pulps, from the Northwest area of Paraná-Brazil, in order to compare the varieties on their enzymatic activity for both, minimum and industrial processing. Enzymatic extracts were prepared from avocado pulp of Choquete, Fortuna and Quintal varieties, in green and ripe maturation stage. Thermal treatment was applied with temperatures 60, 65, 70, 75 and 80 °C. The enzymatic activities were determined by using spectrophotometer. A decline of polyphenoloxidase activity was observed in all of the varieties when both, temperature and time increased. Total inactivation of enzymes was not observed in the largest temperature. Fortuna and Choquete variety showed the lowest polyphenoloxidase activity in the ripe stage. Soluble peroxidase showed activity in the green stage, whereas, ionically bound peroxidase activity increased with the change from green to ripe maturation stage in Choquete variety.

Deoxynivalenol (DON) degradation and peroxidase enzyme activity in submerged fermentation

This work aims to evaluate deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in a submerged fermentation system and to correlate it to the activity of oxydo-reductase enzymes. The submerged medium consisted of sterile distilled water contaminated with 50 μg of DON and 4 × 10(6) spore.mL-1 inoculum of Aspergillus oryzae and Rhizopus oryzae species, respectively in each experiment. Sampling was performed every 24 hours for monitoring the peroxidase specific activity, and every 48 hours for determining mycotoxin levels. Results showed that the fungi species were able to decrease DON levels as the peroxidase activity increased. The 48 hours fermentation interval presented the highest peroxidase specific activity (ΔABS/minute.μg.protein-1), 800 and 198, while the highest DON degradation velocity was 10.8 and 12.4 ppb/hour, respectively in both cases for Rhizopus oryzae and Aspergillus oryzae.

Thermal inactivation of polyphenoloxidase and peroxidase in Jubileu clingstone peach and yeast isolated from its spoiled puree

The thermal inactivation of yeast isolated from spoiled Jubileu peach puree and that of polyphenoloxidase (PPO) and peroxidase (POD) in cv. Jubileu, which is widely cultivated in southern Rio Grande do Sul state, Brazil, were studied. PPO and POD were extracted using the protein powder method and submitted to partial purification by precipitation followed by dialysis. The enzymatic activity was determined measuring the increase in absorbance at 420 nm for PPO and 470 nm for POD. The yeast used in this investigation was isolated from spoiled Jubileu peach puree at 22 °Brix, with total initial microbial count of 22 × 10² UFCmL- 1. Stock cultures were maintained on potato dextrose agar (PDA) slants at 4 °C and pH 5 for later use for microbial growth. In all cases, kinetic analysis of the results suggests that the thermal inactivation was well described by a first-order kinetic model, and the temperature dependence was significantly represented by the Arrhenius law. Both enzymes were affected by heat denaturation, and PPO was more thermostable. PPO was also more thermosTable than the yeast isolated from peach puree. The D60-values were 1.53 and 1.87 min for PPO and yeast isolated from spoiled Jubileu peach puree, respectively.

Peroxidase activity and sensory quality of ready to cook mixed vegetables for soup: combined effect of biopreservatives and refrigerated storage

Enzymatic senescence processes and browning of fresh cut vegetables negatively affect their sensory properties and nutritional value and finally result in the rejection of affected products by consumers. In order to prevent quality decay, the combined effects of natural antioxidants and storage temperature on peroxidase activity and sensory attributes (overall visual quality, browning and odor) of individual and mixed vegetables for soup (butternut squash, leek and celery) were evaluated. Fresh cut vegetables were treated with antioxidant solutions as tea tree essential oil (15 μl/mL), propolis extract (15 μl/mL) and gallic acid (2 mg/mL) and stored at optimal (5 °C) and abusive (15 °C) temperature for a maximum of 14 days. The application of natural preservatives, plus optimal storage conditions, exerted significant inhibitory effects in peroxidase activity of squash, celery and mixed vegetables throughout the storage. Furthermore, propolis treatment applied on mixed vegetables retarded browning appearance and preserved the visual quality for a longer period when compared to untreated product.

Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.

Optimization of Lignin Peroxidase, Manganese Peroxidase, and Lac Production from Ganoderma lucidum Under Solid State Fermentation of Pineapple Leaf

This study was undertaken to isolate ligninase-producing white-rot fungi for use in the extraction of fibre from pineapple leaf agriwaste. Fifteen fungal strains were isolated from dead tree trunks and leaf litter. Ligninolytic enzymes (lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac)), were produced by solid-state fermentation (SSF) using pineapple leaves as the substrate. Of the isolated strains, the one showing maximum production of ligninolytic enzymes was identified to be Ganoderma lucidum by 18S ribotyping. Single parameter optimization and response surface methodology of different process variables were carried out for enzyme production. Incubation period, agitation, and Tween-80 were identified to be the most significant variables through Plackett-Burman design. These variables were further optimized by Box-Behnken design. The overall maximum yield of ligninolytic enzymes was achieved by experimental analysis under these optimal conditions. Quantitative lignin analysis of pineapple leaves by Klason lignin method showed significant degradation of lignin by Ganoderma lucidum under SSF

Contribution of antioxidant enzymes to toxicity assessment in fluvial biofilms

Per tal d’avaluar l’impacte de la contaminació en els ecosistemes aquàtics, aquesta tesi es centra en una aproximació multi-biomarcador en els biofilms. En complement dels biomarcadors clàssics, es va demostrar que les activitats dels enzims antioxidants (AEA): catalasa, ascorbat peroxidasa i glutatió reductasa eren biomarcardors d'estrès oxidatiu en els biofilms. Tot i que les AEA poden veure's influenciades amb la mateixa mesura per factors naturals (edat del biofilm, llum de colonització o d'exposició) i contaminants (herbicides i farmacèutics), aquestes AEA permeten entendre millor l'efecte dels contaminants. Cal remarcar que assajos de toxicitat aguda es poden utilitzar per comparar la capacitat antioxidant entre comunitats i conèixer la seva pre-exposició a l'estrès oxidatiu. Aquesta aproximació multi-biomarcador a nivell de comunitat és especialment interessant per avaluar la toxicitat dels contaminants emergents (β-blockers) sobre espècies no-diana. Per tal de millorar-la, també es va verificar la possibilitat de mesurar l'expressió gènica en biofilms.

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO center dot) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage. (c) 2007 Elsevier B.V. All rights reserved.

Restricted expression of NADPH oxidase/peroxidase gene (Duox) in zone VII of the ascidian endostyle

The ascidian Ciona intestinalis, a marine invertebrate chordate, is an emerging model system for developmental and evolutionary studies. The endostyle, one of the characteristic organs of ascidians, is a pharyngeal structure with iodine-concentrating and peroxidase activities and is therefore considered to be homologous to the follicular thyroid of higher vertebrates. We have previously reported that a limited part of the endostyle (zone VII) is marked by the expression of orthologs of the thyroid peroxidase (TPO) and thyroid transcription factor-2 (TTF-2/FoxE) genes. In this study, we have identified the Ciona homolog of NADPH oxidase/peroxidase (Duox), which provides hydrogen peroxide (H2O2) for iodine metabolism by TPO in the vertebrate thyroid. Expression patterns assessed by in situ hybridization have revealed that Ciona Duox (Ci-Duox) is predominantly expressed in the dorsal part of zone VII of the endostyle. Furthermore, two-color fluorescent in situ hybridization with Ci-Duox and Ciona TPO (CiTPO) has revealed that the ventral boundary of the Ci-Duox domain of expression is more dorsal than that of CiTPO. We have also characterized several genes, such as Ci-Fgf8/17/18, 5HT7, and Ci-NK4, which are predominantly expressed in the ventral part of zone VII, in a region complementary to the Ci-Duox expression domain. These observations suggest that, at the molecular level, zone VII has a complex organization that might have some impact on the specification of cell types and functions in this thyroid-equivalent element of the ascidian endostyle.

Haem peroxidase activity in Daphnia magna: A biomarker for sub-lethal toxicity assessments of kerosene-contaminated groundwater

A novel biomarker was developed in Daphnia magna to detect organic pollution in groundwater. The haem peroxidase assay, which is an indirect means of measuring oxidase activity, was particularly sensitive to kerosene contamination. Exposure to sub-lethal concentrations of kerosene-contaminated groundwater resulted in a haem peroxidase activity increase by dose with a two-fold activity peak at 25%. Reproduction in D. magna remained unimpaired when exposed to concentrations below 25% for 21 days, and a decline in fecundity was only observed at concentrations above the peak in enzyme activity. The measurement of haem peroxidase activity in D. magna detected sublethal effects of kerosene in just 24 h, whilst offering information on the health status of the organisms. The biomarker may be useful in determining concentrations above which detrimental effects would occur from long-term exposure for fuel hydrocarbons. Moreover, this novel assay detects exposure to chemicals in samples that would normally be classified as non-toxic by acute toxicity tests.

Genistein protects human mammary epithelial cells from benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and 4-hydroxy-2-nonenal genotoxicity by modulating the glutathione/glutathione S-transferase system

Epidemiological studies have shown that ingestion of isoflavone-rich soy products is associated with a reduced risk for the development of breast cancer. In the present study, we investigated the hypothesis that genistein modulates the expression of glutathione S-transferases (GSTs) in human breast cells, thus conferring protection towards genotoxic carcinogens which are GST substrates. Our approach was to use human mammary cell lines MCF-10A and MCF-7 as models for non-neoplastic and neoplastic epithelial breast cells, respectively. MCF-10A cells expressed hGSTA1/2, hGSTA4-4, hGSTM1-1 and hGSTP1-1 proteins, but not hGSTM2-2. In contrast, MCF-7 cells only marginally expressed hGSTA1/2, hGSTA4-4 and hGSTM1-1. Concordant to the protein expression, the hGSTA4 and hGSTP1 mRNA expression was higher in the non-neoplastic cell line. Exposure to genistein significantly increased hGSTP1 mRNA (2.3-fold), hGSTP1-1 protein levels (3.1-fold), GST catalytic activity (4.7-fold) and intracellular glutathione concentrations (1.4-fold) in MCF-10A cells, whereas no effects were observed on GST expression or glutathione concentrations in MCF-7 cells. Preincubation of MCF-10A cells with genistein decreased the extent of DNA damage by 4-hydroxy-2-nonenal (150 mu M) and benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (50 mu M), compounds readily detoxified by hGSTA4-4 and hGSTP1-1. In conclusion, genistein pretreatment protects non-neoplastic mammary cells from certain carcinogens that are detoxified by GSTs, suggesting that dietary-mediated induction of GSTs may be a mechanism contributing to prevention against genotoxic injury in the aetiology of breast cancer.