Extraction Of Peroxidase Research Articles

Comparative Analyses of Universal Extraction Buffers for Assay of Stress Related Biochemical and Physiological Parameters

Comparative efficiency of three extraction solutions, including the universal sodium phosphate buffer (USPB), the Tris-HCl buffer (UTHB), and the specific buffers, were compared for assays of soluble protein, free proline, superoxide radical (), hydrogen peroxide (H2O2), and the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) in Populus deltoide. Significant differences for protein extraction were detected via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE). Between the two universal extraction buffers, the USPB showed higher efficiency for extraction of soluble protein, CAT, GR, , GPX, SOD, and free proline, while the UTHB had higher efficiency for extraction of APX, POD, and H2O2. When compared with the specific buffers, the USPB showed higher extraction efficiency for measurement of soluble protein, CAT, GR, and , parallel extraction efficiency for GPX, SOD, free proline, and H2O2, and lower extraction efficiency for APX and POD, whereas the UTHB had higher extraction efficiency for measurement of POD and H2O2. Further comparisons proved that 100 mM USPB buffer showed the highest extraction efficiencies. These results indicated that USPB would be suitable and efficient for extraction of soluble protein, CAT, GR, GPX, SOD, H2O2, , and free proline.

Guaiacol peroxidase zymography for the undergraduate laboratory.

This laboratory exercise presents a novel way to introduce undergraduate students to the specific detection of enzymatic activity by electrophoresis. First, students prepare a crude peroxidase extract and then analyze the homogenate via electrophoresis. Zymography, that is, a SDS-PAGE method to detect enzyme activity, is used to specifically detect peroxidase activity and furthermore, to analyze the total protein profile. After the assay, students may estimate the apparent molecular mass of the enzyme and discuss its structure. After the 4-h experiment, students gain knowledge concerning biological sample preparation, gel preparation, electrophoresis, and the importance of specific staining procedures for the detection of enzymatic activity.

Extraction of peroxidase from waste Brassica oleracea used for the treatment of aqueous phenol in synthetic waste water

In this study, peroxidase was extracted from waste cauliflower stem and leaf and was partially purified. Peroxidase activity of cauliflower stem extract was found to be greater than that in leaf extract. Optimal temperature, pH and ionic strengths for cauliflower stem extract were found to be using pyrogallol/H2O2 substrate and used to catalyze the oxidation and polymerization of phenolic compounds in synthetic waste water. Further, the effect of H2O2, enzyme concentration, initial phenol and polyethylene glycol (PEG) concentrations was investigated on the phenol removal process. In the presence of PEG the phenol removal increased dramatically from 35% to >90% and FT-IR study on phenol samples before and after the peroxidase treatment showed the phenol radical formation and a decrease in phenol concentration.

Three phase partitioning a novel technique for purification of peroxidase from orange peels (Citrus sinenses)

The present work describes the work on three phase partitioning (TPP) of a peroxidase enzyme from orange peels (Citrus sinenses). TPP is a bioseparation technique which has been used for the extraction, concentration and purification of enzymes as well as natural products. Three phase system is formed with simultaneous addition of ammonium sulfate directly to crude broth/feed followed by addition of t-butanol. Effect of different process parameters such as time, ammonium sulfate concentration, pH, broth to t-butanol ratio, temperature and speed of agitation have been studied on purification fold and activity recovery. Maximum purity of 18.20-fold with 93.96% recovery has been obtained with optimized parameters. Optimized system comprised of solution saturated with 50% ammonium sulfate at pH 6 and temperature 30°C with broth to t-butanol ratio 1:1.5 (v/v) with 200rpm with for 80min. SDS PAGE analysis of partially purified enzyme shows molecular weight of 26kDa. TPP was found to be an attractive technique for the extraction and partial purification of peroxidase from orange peels.

Use of hairy roots extracts for 2,4-DCP removal and toxicity evaluation by Lactuca sativa test.

2,4-Dichlorophenol (2,4-DCP) is widely distributed in wastewaters discharged from several industries, and it is considered as a priority pollutant due to its high toxicity. In this study, the use of different peroxidase extracts for 2,4-DCP removal from aqueous solutions was investigated. Tobacco hairy roots (HRs), wild-type (WT), and double-transgenic (DT) for tomato basic peroxidases (TPX1 and TPX2) were used to obtain different peroxidase extracts: total peroxidases (TPx), soluble peroxidases (SPx), and peroxidases ionically bound to the cell wall (IBPx). All extracts derived from DT HRs exhibited higher peroxidase activity than those obtained from WT HRs. TPx and IBPx DT extracts showed the highest catalytic efficiency values. The optimal conditions for 2,4-DCP oxidation were pH 6.5, H2O2 0.5 mM, and 200 U mL(-1) of enzyme, for all extracts analyzed. Although both TPx extracts were able to oxidize different 2,4-DCP concentrations, the removal efficiency was higher for TPx DT. Polyethylene glycol addition slightly improved 2,4-DCP removal efficiency, and it showed some protective effect on TPx WT after 2,4-DCP oxidation. In addition, using Lactuca sativa test, a reduction of the toxicity of post removal solutions was observed, for both TPx extracts. The results demonstrate that TPx extracts from both tobacco HRs appear to be promising candidate for future applications in removing 2,4-DCP from wastewaters. This is particularly true considering that these peroxidase sources are associated with low costs and are readily available. However, TPx DT has increased peroxidase activity, catalytic efficiency, and higher removal efficiency than TPx WT, probably due to the expression of TPX1 and TPX2 isoenzymes.

Extraction and characterization of peroxidase from Camellia sinensis

The state Assam located in the eastern part of India is well known in the world as a major tea grower. Peroxidase is an oxidative enzyme known to be involved in defence reactions in plants. Peroxidase enzyme extracted from Camellia sinensis (tea) leaves collected from 5 leading tea estates located in district Dibrugarh of Assam, India was characterized and optimization study was carried out. Its effect on varying substrate concentration, temperature, pH and salt was studied. Its activity was found to be inhibited by ethylene-diamine-tetra-acetic acid. The optimum temperature and pH was 30 °C and 6.5 respectively. The enzyme was thermostable showing activity up to 60 °C. Its specific activity was 6.19 U/mg protein and km and Vmax values were 0.01 mg/mL (0.04 mM) and 0.03 mg/mL (0.12 mM) respectively. With all the above features, the enzyme peroxidase is proved to be useful for industrial purposes.

Crude peroxidase from onion solid waste as a tool for organic synthesis. Part III: synthesis of tetracyclic heterocycles (coumestans and benzofuroquinolinones)

In the present study the biocatalytic activity of a crude onion peroxidase extract on the reaction between catechol and heterocyclic 1,3-dicarbonyl compounds is investigated. The crude enzyme preparation promotes effectively the reaction between catechol and 4-hydroxycoumarin, 4-hydroxy-1-methyl-2-quinolinone and 2,4-quinolinediol to afford the corresponding coumestans and benzofuroquinolinones in satisfactory yields. The products of the reaction are fully characterized spectroscopically and are found to possess potent antioxidant activity as evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and luminol-induced chemiluminescence assays.

Obtention of plant peroxidase and its potential for the decolorization of the reactive dye Remazol Turquoise G 133%

Peroxidases can be used in the decolorization process. There is a growing interest for new sources of this enzyme and for obtaining economically viable processes. In this work, a low-cost vegetable peroxidase extraction process is proposed; the resulting enzyme is characterized to determine its optimum pH, temperature, and stability conditions, and it is then applied in the decolorization of reactive dye Remazol Turquoise G 133%. The turnip peroxidase (TP) was utilized as an enzymatic source. This enzyme exhibited maximum activity at pH 7.0, and it was active in the temperature range of 30 to 50 °C, which favors its use in industrial processes. Acetone was the most efficient solvent to induce precipitation. The removal of Remazol Turquoise G 133% was 56.0% complete after 50 min, while 41.0% of the same dye was removed with the commercial horseradish peroxidase enzyme in 50 min. TP presents potential as a viable alternative in the decolorization of textile wastewaters.

Modulation of endogenous peroxidase by exogenous peroxidase in chinese red radish seedling

To investigate the peroxidase function and its signal pathway, activity and isozyme pattern of peroxidase and transcription level of the peroxidase gene rsprx1 and rsprx2 in Chinese red radish seedling (Raphanus sativus L.) were investigated after administration of the exogenous peroxidase (extract of radish). The activity and isozyme pattern of peroxidases were increased, among cotyledon, hypocotyl and root, the most significant increase of peroxidases activity was observed in hypocotyls, the activity of phenylalanine ammonialyase was also increased, but the pelargonidin content was decreased. Although the exogenous application of horseradish peroxidase and the extract of radish could both affect the activity of endogenous enzyme and metabolites of Chinese red radish seedlings, the latter was more effective than the former. Nifedipine, as L-calcium channel blocker, affected the activity of peroxidase and pelargonidin content in Chinese red radish seedling. Exogenously added nifedipine with the extract caused reversal of the effects. It is concluded that calcium was involved in the signal pathway of the extract of radish in Chinese red radish seedling.

One-pot synthesis and characterization of cross-linked quaternized chitosan microspheres as protein adsorbent

The one-pot synthesis and characterization of cross-linked quaternized chitosan microspheres (CQCM) as a protein adsorbent are presented. First of all, chitosan particles were prepared by spray drying method, and then they were quaternized and cross-linked in turn with glycidyltrimethylammonium (GTMAC) chloride and glutaraldehyde in isopropanol containing 10% water in one-pot. The effect of the reaction temperature, reaction time and the amounts of added GTMAC and glutaraldehyde on the protein adsorption ability of CQCM was investigated. The adsorption behavior of the CQCM prepared in the optimum synthetic conditions was well described by the Langmuir isotherm with maximum adsorption capacity equal to 1424mg BSA/g dry weight. The particle size ranged from 7.6 to 48.9μm. The mechanism of adsorption–desorption of BSA to the CQCM was ion-exchange. Finally, the extraction of soybean peroxidase from crude soybean peroxidase solution using the CQCM was performed.

Peroxidase and polyphenoloxidase activity in tomato in natura and tomato purée

Oxidative enzymes are responsible for changes in flavor, texture, color and also in some nutritive properties of several fruits and vegetables processed in industries. The present investigation aimed to study the activity of Peroxidase (E.C. 1.11.1.7) and Polyphenoloxidase (E.C. 1.10.3.1) in tomato fresh pulp in natura and in three different trademarks of tomato puree sold in supermarkets. Crude extracts of peroxidase (POD) and polyphenoloxidase (PPO) enzymes, from both fresh tomato pulp in natura and processed tomato puree, purchased in supermarkets, were prepared, by using sodium phosphate buffer at different pHs. The best pH values for extraction of peroxidase and polyphenoloxidase were 6.2 and 7.5, respectively. Protein precipitations were carried out using acetone at the ratio of 1:2 enzymatic extract/acetone. The enzymatic extract from Santa Clara tomato cultivar was concentrated 10 times, before being applied to the chromatographic column packed with Sephacryl 100-HS. From the samples eluted, one isoenzyme was isolated (A1) and the molecular weight and isoelectric point (pI) was determined (MW 45 kDa and pI 4.5). The fraction with A1 was stored at -18 o C, and later used for thermostability studies. A decrease in the enzymatic activity was observed at 85 and 90C, in the enzymatic extracts obtained from fresh tomato pulp; nevertheless, in the processed tomato purees the loss of activity was more significant.

Removal of olive mill waste water phenolics using a crude peroxidase extract from onion by-products

Olive mill waste water (OMWW) originating from a two-phase olive oil-producing plant was treated with a crude peroxidase extract prepared from onion solid by-products. The treatments were based on a 3 × 3 factorial design, employing a series of combinations of pH and H2O2, in order to identify optimal operational conditions. The treatment performance was assessed by estimating the removal percentage of total polyphenols. The model established produced a satisfactory fitting of the data (R2 = 0.94, p = 0.0158), while the second-order polynomial equation used to describe the process indicated that peroxidase-catalysed polyphenol removal in diluted OMWW is facilitated at relatively low pH and intermediate H2O2 values. A predicted value of 50.7 ± 9.5% removal was calculated under optimal operational conditions (pH 2.76, [H2O2] = 3.56 mM). Analysis of an untreated and an optimally treated sample by high performance liquid chromatography revealed that enzyme treatment brought about alteration in the original OMWW polyphenolic profile. The use of peroxidase from onion solid by-products is proposed as an alternative means that could have a prospect in bioremediation applications.

Palmito de pupunha (Bactris gasipaes Kunth.) composição mineral e cinética de enzimas oxidativas

A análise da presença de enzimas oxidativas como a peroxidase (POD) e a polifenoloxidase (PPO) e o controle da atividade destas enzimas são importantes na preservação e no processamento de alimentos. Este trabalho teve por objetivo determinar a atividade enzimática da polifenoloxidase (PPO) e da peroxidase (POD) do palmito de pupunha, bem como avaliar o comportamento destas enzimas frente ao tratamento térmico e assim calcular a cinética de inativação térmica das mesmas para suas porções termorresistente e termolábil. Para a extração de peroxidase (POD) e polifenoloxidase (PPO) de palmito, utilizou-se solução tampão fosfato de sódio 100 mM com diferentes pHs (5,5; 6,0; 6,5 e 7,0). O melhor pH de extração da POD foi 5,5 e da PPO, 6,5. Estes extratos foram tratados em diferentes temperaturas (65, 70, 75 e 80 °C) por períodos de 1 a 10 minutos. A POD e a PPO sofreram um decréscimo de 70 e 80%, respectivamente, em relação às suas atividades iniciais. As energias de ativação, nas temperaturas estudadas, para a porção termolábil e termorresistente da peroxidase foram 154,0 e 153,0 kJ.mol-1, respectivamente, enquanto que para a polifenoloxidase foram 26,3 e 27,0 kJ.mol-1, respectivamente. Resultados apresentaram valores que estão dentro da faixa de energia de ativação reportada para o processo de inativação térmica de enzimas.

Extracción y medida de peroxidasa en pulpa de arazá (Eugenia stipitata MC Vaugh)

A technique for both extraction and activity measurement of peroxidase extracted from araza (Eugenia stipitata Mc Vaugh) is described. Peroxidase from araza pulp fruit was extracted using a combination of protein precipitation with acetone and extraction with 50 mM sodium buffer phosphate (pH 6.0). Optimum activity using guaiacol as H-donor was obtained at pH from 5.0 to 6.5, temperature from 60 to 75 °C, H2O2 between 10 to 15 mM and guaiacol from 80 to 160 mM. Thermal inactivation showed a first-order inactivation kinetic. Reactivation was observed when extracts were heated at 80 °C and afterwards incubated at 25 °C.

Characterization and Role of Polyphenol Oxidase and Peroxidase in Browning of Fresh-Cut Melon

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from two different varieties of melon ( Cucumis melo L. cantalupensis cv. Charentais and C. melo L. inodorus cv. Amarillo) and characterized using reliable spectrophotometric methods. In both cases the enzymes followed Michaelis-Menten kinetics, showing different values of kinetics parameters between the two cultivars: K m = 7.18 +/- 0.70 mM ('Charentais') and 6.66 +/- 0.20 mM ('Amarillo') mM; V max = 7.93 +/- 0.35 units/min ('Charentais') and 13.82 +/- 0.37 units/min ('Amarillo'), relative to PPO; K m = 24.0 +/- 2.10 mM ('Charentais') and 5.05 +/- 0.19 mM ('Amarillo') mM; V max = 344.83 +/- 10.32 units/min ('Charentais') and 80.64 +/- 2.01 units/min ('Amarillo'), relative to POD. Optimum pH for PPO was 7.0 for 'Charentais' and 7.5 for 'Amarillo, whereas it was 4.5 for both cultivars relative to POD. Melon PPO had maximum activity at 60 degrees C in both 'Charentais' and 'Amarillo' cultivars, whereas POD maximum activity was found at 45 degrees C in 'Charentais' and at 25 degrees C in 'Amarillo'. POD from both cultivars showed higher thermolability compared with PPO, losing >90% of relative activity after only 5 min of incubation at 70 degrees C. POD's activation energy was much higher than that of PPO (Delta E (#) = 86.3 and 160.6 kJ mol (-1) for 'Charentais' and 'Amarillo', respectively). PPO and POD activities from both cultivars showed a decreasing pattern as sugar concentration in the assay medium increased, except in POD extract from 'Charentais', which maintained its activity in the presence of high d-glucose concentration (up to 5 M). Changes in L*, a*, b*, chroma, and hue angle values were chosen to describe the browning development in the samples during storage at 5 degrees C. A slight decrease in L* value and a more marked reduction of a* value were noted in both cultivars above all at the end of storage period. POD activity during storage at 5 degrees C was highly correlated with changes of parameters a*, b*, chroma, and hue angle ( r (2) from 0.82 to 0.97) for cultivar 'Charentais'. According to these results, only POD activity seemed to be involved in browning of minimally processed melon.

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 °C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to LiP of Phanerochaete chrysosporium and a total inhibition of LiP was observed.

Reação enzimática ao estresse salino durante a germinação de estilosantes

O objetivo deste trabalho foi avaliar a influência do NaCl nos sistemas isoenzimáticos durante o estádio inicial de germinação do Stylosanthes guianensis (Aubl.) Sw. Sementes das variedades botânicas canenscens, microcephala, pauciflora e vulgaris foram colocadas para germinar em caixas de plástico, com papel-filtro umedecido com água (controle) e solução de NaCl (134 mmol), em estufa BOD, por 14 horas. Após este período, foi realizada a extração das enzimas glutamato desidrogenase e peroxidase. As enzimas apresentaram comportamentos diferenciados nas quatro variedades quando submetidas ao estresse salino.

Ecophysiological approach to study of the role of cell-bound peroxidase forms in wheat leaves of different morphology

Extracellular peroxidase enzymes, both ionically - and covalently bound, have been implicated in the biochemical changes of cell wall and furthermore for regulation of leaf and root extension growth. However, the role of cell wall bound peroxidase in changes of mature leaf morphology is poorly understood. The aim of this study was to determine whether ionically and covalently bound peroxidase activities exhibit any relationships with drought-induced leaf rolling of wheat. An activity and thermostability of three wheat genotypes with different leaf rolling degree were studied. The results clearly show that two genotypes with leaf rolling trait exhibited significantly higher activity of both extracellular peroxidase fractions in comparison to non-rolling cv. 'Omskaya 9'. In addition, the significant genotypic differences were found in thermostability of ionically and covalently bound peroxidase. A 35 °C-treatment decreased the activity of ionically bound peroxidase (IBP) in non-rolling leaves of parent cultivar and enhanced the activity of IBP in rolling leaves of 'Alba' genotype. Under 45 and 55 °C-treatment the IBP was more stable in rolling leaves of both genotypes 'Otan' and 'Alba'. Despite to low activity of covalently bound peroxidase it was more stable after heating by 45 °C compare with ionically bound fraction. The heating of covalently bound peroxidase (CBP) extracts by 35 °C and 45 °C also has enhancing effect on its activity by both leaf rolling genotypes (on 16 and 40% by 'Otan' and 'Alba', respectively). At the same time the CBP activity of non-rolling genotype decreased significantly on 40%. Even after 55 °C-treatment the CBP of both rolling genotypes remained very stable, although there were further inhibition of CBP purificated from leaves of non-rolling genotype. Both wheat genotypes with inserted leaf rolling trait exhibited a much higher blue-green fluorescence emission than non-rolling leaves of parent cultivar 'Omskaya 9'. Thus, the molecular mechanism of leaf rolling regulation seems to include an adjustment of cell wall properties and probably involves an activity of cell wall bound peroxidase. Further investigation is expected to reveal the regulatory mechanism for leaf rolling in wheat.

Mass transfer in a spray column during two-phase extraction of horseradish peroxidase

Multistage contactors are often needed to achieve the desired degree of mass transfer. One such contactor is spray column, which can be conveniently used to isolate and purify proteins using aqueous two-phase extraction. The effects of orifice size and addition of NaCl on the overall mass transfer coefficient ( K L a) of horseradish peroxidase (HRP) and fractional dispersed phase hold up ( ε D) have been studied in a simple spray column using a polyethylene glycol/potassium phosphate aqueous two-phase system. K L a and ε D increased with an increase in dispersed phase superficial velocity ( v s) as well as capillary diameter. The presence of NaCl increased K L a. The overall mass transfer coefficient for HRP normalized for dispersed phase hold up, was found to be independent of v s in the range studied.

Involvement of Blueberry Peroxidase in the Mechanisms of Anthocyanin Degradation in Blueberry Juice

ABSTRACT: Addition of hydrogen peroxide (H2 O2) to blueberry juice changed the red coloration toward brown. Addition of ascorbic acid prevented the formation of brown polymers. An extract of peroxidase (POD) prepared from blueberry fruits was able to oxidize CG into the corresponding o‐quinone but only in the presence of H2 O2. The chlorogenoquinone plays a dominant role in anthocyanin degradation. We demonstrated that peroxidase extract in the absence of CG showed a weak degradation activity toward blueberry anthocyanins, cyanidin 3‐glucoside, and pelargonidin 3‐glucoside. Nevertheless, addition of CG increased anthocyanin degradation, leading to formation of brown polymers. Therefore, blueberry POD could participate in the development of browning during blueberry‐juice storage.