Peroxidase activity in Spondias dulcis/Atividade da peroxidase em Spondias dulcis.
Spondias dulcis (caja-mango) is a small fruit cultivated in the northeast region of Brazil, mainly during the rainy season. As with most regional fruits, caja-mango is available during a short period of the year, and rarely found in other regions of the country, and it is not found in foreign countries (HAMANO; MERCADANTE, 2001).
Spondias spp. are genera members of the family Anacardiaceae. This genus is widespread in tropical regions of the world. Tropical fruits have found a firmly growing acceptance in the markets of Europe and United States. This increasing interest may be related to the public's nutritional awareness and search for a healthier lifestyle, as well as for their exotic flavors (CEVA-ANTUNES et al., 2003; CEVA-ANTUNES et al., 2006; LIRA JUNIOR et al., 2005; NARAIN et al., 2004).
Brazil has a natural abundance of tropical fruits with distinctive
flavors appealing to the foreign consumer (FRANCO; SHIBAMOTO, 2000). In some regions of the country exploitation of natives fruits is responsible for a large parcel of employment (CEVA-ANTUNES et al., 2006).
In general, vegetables are very susceptible to undesirable alterations as a consequence of injuries suffered during storage, handling and processing. The most usual alterations are changes in texture, nutritive qualities, color and flavor, which decrease the market value of the product (ESCRIBANO et al., 2000; VALDERRAMA et al., 2001). An important cause of color alterations is due to either formation or degradation of pigmented compounds usually present in the product. This process of deterioration is mediated by endogenous enzymatic activities such as polyphenol oxidase (PPO) and peroxidase (POD), which leads to the formation of dark polymers of a quinoidal nature (ESCRIBANO et al., 2000; FRICKS et al., 2006; PEREZ-TELLO et al., 2001; VALENTINES et al., 2005; ZHOUA et al., 2003).
Peroxidases (EC 220.127.116.11.) are members of a large group of enzymes widely distributed in nature, the oxidoreductases, which are involved in the oxidation of different substrates at the expense of hydrogen peroxide (KHAN; ROBINSON, 1993a; SANTOS et al., 2003, 2004). Generally, plant peroxidases have a specific requirement for hydrogen peroxide and may be considered as peroxide scavengers (KHAN; ROBINSON, 1993b). These enzymes have been implicated in metabolic process such as ethylene biogenesis, cell development and membrane integrity (SILVA et al., 1990).
Peroxidases are recognized as being one the most heat-stable enzymes present in vegetables. Then, inactivation of peroxidase activity is usually used to indicate the adequacy of blanching treatments (AGUERRO et al., 2005; KHAN; ROBINSON, 1993a; McLELLAN; ROBINSON, 1984; ZANATTA et al., 2006). The high thermal resistance of peroxidases and their ability to undergo reactivation call for a study of the thermal stability of such enzymes during the blanching of fruits and vegetables (PHILIPPON; ROUET-MAYER, 1984).
Therefore, the quality of the blanched and frozen products may be better if there is some activity of peroxidase left at the end of the blanching process (GARROTE et al., 2004). A residual activity of peroxidases of about 3-10% after blanching was recommended for preservation by freezing (GUNES; BAYUNDIRH, 1993). Blanching time would be the time required to achieve the inactivation of peroxidase and it depends on such factors as heating method, heating temperature, size, shape and thermal conductivity of the product and type and concentration of the enzymes. This multiplicity of concurrent factors makes it usual that the blanching time is established empirically (AGUERRO et al., 2005; DENG et al., 2003; LEMOS et al., 2000; SERVANT et al., 1986a; SERVANT et al., 1986b).
The aim of the present work was to determine the initial conditions for purification and characterization of S. dulcis peroxidase, and to estimate conditions for the inactivation of the POD.
Material and methods
Plant material. 500 g of Spondias dulcis fruits were obtained in the Rio de Janeiro city market in September 2006. The samples were stored in a freezer.
Enzyme extraction. 25 g of fresh pulp was blended in different sodium phosphate buffer ranging pH 3.0 to 9.0 (Table 1) and the extract was centrifuged at 23000 x g for 20 min. at 4[degrees]C (twice). The supernatant (raw extract) was collected and assayed for soluble peroxidase activity (FRICKS et al., 2006).
Precipitation of proteins. The proteins were precipitated from supernatants by adding solid ammonium sulfate to reach up to 90% (w [v.sup.-1]) of saturation. Next the solution was centrifuged at 2300 x g for 40 min. at 4[degrees]C, the suspension was discarded. The precipitate was dissolved in a minimal volume of phosphate buffer 0.2 M (pH 6.5) (FRICKS et al., 2006).
Determination of the peroxidase activity.
Enzyme activity was determined measuring the change in absorbance at 470 nm due to the formation of the oxidation product of guaiacol, described to be tetraguaiacol ([[epsilon].sub.tetraguaiacol] = 2.66 x [10.sup.4] [M.sup.-1] [cm.sup.-1]) (HIRATA et al., 1998), in the presence of hydrogen peroxide and the enzyme (GORANLL et al., 1949). One unit of enzyme (U) is defined as the quantity of enzyme able of forming 1 [micro]mol of chemical product per minute.
Protein determination. Protein concentrations of the extracts were measured according to the Bradford (1976) method, measuring optical density (OD) at 595 nm, with bovine serum albumin as standard.
Effect of pH. A study was made on the effect of pH on guaiacol oxidation by S. dulcis peroxidase. Enzyme activity was determined in 0.2 M sodium phosphate buffer at different pH values, ranging from 5.0 to 8.0.
Heat-inactivation. Heat-inactivation studies of peroxidase activity were carried out on the proteins precipitated in the test tubes. Following heat inactivation, the tubes were immersed in cold water to minimize any further change in enzymatic activity. By this method, it is determined the residual activity of the enzyme present in treated solutions.
Results and discussion
Determination of suitable conditions for peroxidase extraction. Several pH and concentration of sodium phosphate buffer were employed to select the most suitable ones to extract peroxidase from S. dulcis tissues. The optimum extraction pH was measured by extracting the peroxidase enzyme from fruits in buffers ranging from pH 3.0 to 9.0 (Figure 1). Maximum peroxidase activity (61 U mg-1) was detected at pH 6.5, with guaiacol as substrate. Moreover, pH values under 5.0 showed low enzyme activity. This effect has been attributed to the loss of the heme group at low pH as described by Pomar et al. (1997). However, at this stage of the work all enzyme assays were undertaken at pH 6.0, due to the best solubility of the enzyme extract aliquots in the reaction mixture.
[FIGURE 1 OMITTED]
Nevertheless, increasing the molar concentration of sodium phosphate buffer the activation of peroxidase is affected (Table 1). It was determined that a 0.2 M concentration of sodium phosphate increases peroxidase activity.
pH optimization for activity assay. Using the optimized extraction conditions with a 0.2 M sodium phosphate buffer, pH 6.5, a study was performed to determine the optimal pH values for reaction of the enzyme (Figure 2). Maximum peroxidase activity was assigned at pH 7.0 (specific activity 106 U [mg.sup.-1] protein).
[FIGURE 2 OMITTED]
Efficiency of precipitation of the raw extract protein. A summary of the activities of the peroxidase (Table 2) shows that the ammonium sulfate fractionation of the raw protein extracts of fruits was efficient, as shown by an increase (13%) of the specific activity and good recovery (66.6%) of the enzymatic activity.
Heat-inactivation. Thermostability plot (Figure 3) shows the residual activities (in per cent) of peroxidase at the protein solution obtained after precipitation of raw extract.
[FIGURE 3 OMITTED]
The inactivation of peroxidase was non-linear with heating time. Khan and Robinson (1993b) reported that the exact cause of the non-linear plots for heat-inactivation is not know, but inactivation is normally a function of pseudo first-order kinetic. At 60[degrees]C the enzyme is stable and activity decreases by 40% after 15 min. But, is has been suggested that this behavior is likely to be due to the presence of a number of iso-peroxidases with different thermostabilities. The thermostability of peroxidases has been attributed to the presence of sugars at their structure (POMAR et al., 1997). The thermal treatment was efficient to total inactivation of peroxidases, enzyme activity extending up to 10 min. After 15 min. at 75[degrees]C the S. dulcis peroxidase maintains 5% of the total activity. Partial regeneration of peroxidase activity was not observed after treatment at 75[degrees]C for 15 min. If the parameter is blanching, Gunes and Bayundirh (1993) reported that a residual activity of peroxidases about 3-10% after this treatment suggest efficiency. However, total inactivation was not achieved which suggests the presence of thermo resistant isoenzymes (ZANATTA et al., 2006). Other studies must be performed with the purpose to clarify the influence of thermal treatment in enzymatic behavior in this fruit.
Maximum peroxidase activity was extracted at pH 6.5 in the sodium phosphate buffer 0.2 M. In this work the enzyme activity assays were conducted at pH 7.0, with guaiacol as substrate.
The results to thermal inactivation of partially purified caja-mango fruit peroxidase suggest that the heating at 75[degrees]C for 15 min. is efficient to inactivate 95% of initial POD activity.
Financial support from Finep, Capes and CNPq is gratefully acknowledged. Special thanks "in memoriam" to Prof. Dsc. Octavio Augusto Ceva Antunes for the orientation in all investigation work, friendship and dedication.
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Received on January 29, 2009.
Accepted on October 14, 2009
License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Dayse Pereira Barbosa Souza (1), Ingrid Chastinet Ribeiro Costa (1), Lucia Moreira Campos Paiva (1), Enrique Guillermo Oestreicher (1), Marcio Eduardo Berezuk (2) and Lucio Cardozo-Filho (2) *
(1) Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil. (2) Departamento de Engenharia Quimica, Universidade Estadual de Maringa, Av. Colombo, 5790, 87020-900, Maringa, Parana, Brazil. * Author for correspondence. E-mail: email@example.com
Table 1. Effect of buffer concentration on S. dulcis peroxidase activity. Specific Activity Buffer Composition (pH = 6.5) (U [mg.sup.-1] protein) 0.2 M sodium phosphate 50 0.1 M sodium phosphate 25 0.05 M sodium phosphate 24 Table 2. Peroxidase activity of the S. dulcis. Raw [N[H.sub.4] Parameter extract [(S[O.sub.4]).sub.2]] 90% saturation Volume (mL) 43 8 Total Protein [mg] 7.3 4.1 Enzymatic activity [U [mL.sup.-1] 1.4 10 Specific activity [U [mg.sup.-1]] 50 57 Total activity [U] 175 116 Recovery [%] 100 67