J. Agr. Sci. Tech. (2016) Vol. 18: 1187-1195 Characterization of Polyphenol Oxidase and Peroxidase From Iranian Medlar ( Mespilus germanica L.) Fruit M. Yolmeh 1, and A. Sadeghi Mahoonak 1* ABSTRACT In this study, the crude protein extract containing PolyPhenolOxidase (PPO) and Peroxidases (POD) were extracted from medlar fruit ( Mespilus germanica L.) grown in Golestan Province, Iran. POD and PPO activities were studied using guaiacol and catechol as substrates, respectively. The effect of pH, temperature and thermal stability, inhibitors and cations were investigated. Results showed that Vmax was higher for PPO compared to the POD. The optimum pHs for POD and PPO were obtained at 6.5 and 5.5, respectively. The optimum temperature for both enzymes was 35°C. The Iranian medlar POD was more thermal stable than the PPO. Ascorbic acid had the highest inhibitory effect on both enzymes. Ca 2+ and Zn 2+ had the highest decreasing and increasing effect on both enzymes. Keywords: Characterization, Medlar, Peroxidase, Polyphenoloxidase. INTRODUCTION Enzymatic browning is a main problem in a number of fruits and vegetables such as Peroxidase (POD, EC 1.11.1.7) are plant potato (Lee and Park, 2007), lettuce et al hemoproteins and oxidoreductase which (Gawlik-Dziki ., 2007) and strawberry et al., catalyze a reaction in which hydrogen (Chisari 2007) which leads to peroxide dose is used as the acceptor and rejection by the consumer. This fact is another substance dose as the donor of caused by conversion of phenolic hydrogen atom. POD is directly involved in compounds to o-quinones, which many plant functions such as hormone subsequently polymerize to be a brown et al., regulation, defense mechanisms, indolacetic pigment (Jiang 2004). In addition, degradation and lignin biosynthesis enzymatic browning may alter plant proteins and be more toxic to potential Downloaded from jast.modares.ac.ir at 13:09 IRST on Thursday September 30th 2021 (Serrano- Martínez et al ., 2008). Moreover, POD is concerned with enzymatic browning phytopathogens (Aydemir, 2004). POD and because diphenols may function as reducing PPO are involved enzymes in the reaction, substrate in enzymatic browning (Chisari et therefore, they have been characterized in al ., 2007). several plants such as the Chinese cabbage Plant polyphenoloxidases (PPO, EC (Nagai and Suzuki, 2001), hot pepper 1.14.18.1) are a group of copper-containing (Arnnok et al ., 2010), potato (Marri et al ., et al enzymes that catalyze oxidation of 2003), broccoli (Gawlik-Dziki ., 2007), polyphenolic compounds which are hot chilli pepper (Schweiggert et al ., 2006), responsible for enzymatic browning thymus (Dogan and Dogan, 2004), and pear et al reactions occurring during various stages of (Hwang ., 1996). processing plant materials (Sheptovitsky and Medlar, Mespilus germanica L., is a Brudwig, 1996). member of Rosaceae, known with local _____________________________________________________________________________ 1 Department of Food Science, Faculty of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Islamic Republic of Iran. Corresponding author; e-mail: [email protected] 1187 _______________________________________________________ Yolmeh and Sadeghi Mahoonak names “Conos ˮ or “Condos ˮ in North of was homogenized in 100 ml of 50 mM cold Iran. The fruit is brown, ranging from 1.5 to acetate baffer (pH 5.5), containing 20 ml of 3 cm in diameter and consumed fresh as 4% Triton X-114, 1 mM MgCl 2, 1 mM well as in jams, jellies and marmalades PhenylMethylSulfonylFuoride (PMSF) as (Bignami, 2000). The native form of medlar protease inhibitor, 2 mM EDTA, for 2 is widely found at forests in Golestan and minutes. The homogenate was filtered Mazandaran Provinces. The Iranian medlar through Whatman No. 42 filter paper and fruits are harvested every year through kept for 1 hour. to complete separation and December and January. The fruit has several followed by centrifuging at 20,000×g for 30 medical benefits such as elimination of minutes. The supernatant was filtered kidney and bladder stones, constipation through Whatman No. 42 filter paper and therapy and as a diuretic (Ayaz et al ., 2008). the supernatant containing protein extract Aydin and Kadioglu (2001) reported total was collected and stored at –20°C until use. soluble sugar, ascorbic acid contents and It was used as a crude enzyme extract for PPO activity of Turkish medlar and Dincer POD and PPO analyses. All steps of enzyme et al . (2002) studied characterization of PPO extraction were performed at 4°C (Ayaz et in Turkish medlar. However, there is not al ., 2008). enough study about the chemical composition of the Iranian medlar fruits. Protein Determination Thus in this paper characterization of POD and PPO from Iranian medlar fruit was studied in terms of optimum pH and Soluble protein content of the extracts was stability, optimum temperature and stability, measured according the method depicted by substrate specificities, degrees of inhibition Gawlik-Dziki et al . (2007), with bovine by general enzyme inhibitors and effect of serum albumin as the standard. metal ions in order to help predict the behavior of Iranian medlar fruit POD and Enzyme Assays PPO. PPO activity was spectrophotometrically MATERIALS AND METHODS measured using the method described by Dincer et al . (2002) with some modifications Medlar fruits ( Mespilus germanica L) and using an ultraviolet-visible UV-160A were harvested from forested regions of spectrophotometer (Shimadzu, Japan) Downloaded from jast.modares.ac.ir at 13:09 IRST on Thursday September 30th 2021 Gorgan, Iran. The fruits were immediately equipped with a quartz cell of 1 cm length. brought to the laboratory and stored at - The activity was measured by calculating 20°C. All chemicals and reagents used were the increase in absorbance at 500 nm and pH analytical grade and purchased from Merck, 7. The reaction mixture consisted of Germany. phosphate buffer solution pH 7 (0.1M, 1.95 mL), 1 mL of 0.1M catechol as a substrate and 50 µL of the enzyme extract. The Enzyme Extraction absorbance at 500 nm was recorded continuously at 25°C for 5 minutes. The The enzymes from Iranian medlar fruits blank sample was also considered containing were extracted using the method described the same mixture solution without the by Ayaz et al . (2008) with some enzyme extract (Dincer et al ., 2002). modifications. Briefly, the samples were POD activity was spectrophotometrically powdered by a grinder (Model 160 determined at 470 nm using guaiacol as a Specimen Grinder, 18 mm diameter) and phenolic substrate with hydrogen peroxide. followed by 50 g the powdered medlar fruit The reaction mixture contained 2.66 mL of 1188 Polyphenol Oxidase and Peroxidase of Medlar ___________________________________ 0.1 M phosphate buffer pH 7, 0.15 mL of the optimum pH values of the substrates, 4% (v/v) guaiacol, 0.15 mL of 1% (v/v) before the addition of the enzyme solution. H2O2, and 40 µL of the enzyme extract. The The relative activity of PPO and POD were blank sample was considered as well spectrophotometrically measured for each (Sahhafi et al ., 2012). temperature by rapid addition of enzyme One unit of PPO and POD activity was extract to the mixture. In order to determine defined as the amount of enzyme that caused the thermal stability of the enzymes, the a change in absorbance of 0.001 min -1 enzyme solution in 50 mM phosphate buffer, (Galeazzi and Sgarbieri, 1981). All the pH 6.5 (because at this pH, the enzymes had experiments were carried out in triplicates high activity), was incubated in a water bath and corresponding means were plotted. at temperatures of 30, 40, 50, 60 and 70°C for 10, 20, 30, and 40 minutes. After the Effect of Substrate Concentration mixture was cooled to room ambient temperature, 0.05 ml heated enzyme extract was mixed with the mixtures of substrate The POD and PPO activity were assayed solution, and the enzymes activity residuals by mixing the enzymatic extract with their were spectrophotometrically measured. The substrate at the different final residual percentage of PPO and POD concentrations, 2, 5, 10, 20, 30, 40, 50, and activity was calculated by comparison with 60 mM. The kinetic results were plotted as unheated respective enzyme (Gawlik-Dziki 1/specific activity (1/V) versus 1/substrate et al ., 2007). concentration (1/[S]). The maximum Velocity (V ) and Michaelis–Menten max Effect of Inhibitors constant (K m) indexes were measured with variable substrate concentrations in the reaction mixture. Substrate specificity Inhibition of enzyme by sodium azide (0- (V max /K m) was measured using the data 10 mM), sodium cyanide (0-10 mM) and obtained on a Lineweaver– Burk plot (Guo ascorbic acid (0-5 mM) were measured. et al ., 2009). Since in pretests was found that ascorbic acid have high inhibitory activity than the Effect of pH other compounds and therefore a lower concentration was used from it. Percent activity graphs were drawn from these The activity of PPO and POD was triplicate results for each inhibitor to find Downloaded from jast.modares.ac.ir at 13:09 IRST on Thursday September 30th 2021 measured using 0.1M citrate buffer (pH 3.5- IC 50 values, which show 50% inhibition of 5), 0.1M phosphate buffer (pH 6-8) and 0.01 the enzyme. M Tris-HCl (pH 8-9.5). The optimum pH for the PPO and POD was obtained using catechol and guaiacol as substrates, Effect of Metal Ions respectively (Ayaz et al ., 2008). Na +, K +, Zn 2+ , Ni 2+ , Ca 2+ and Fe 3+ were used Effect of Temperature as metal ions to measure POD and PPO activity. The concentrations of each metal ion in the enzyme assay were 0.2 and 0.5 The PPO and POD activity was measured mM. The concentrations were used giving at various temperatures controlled by a the performed pretests.