Partial Purification and Characterisation of Polyphenol Oxidase from Two Commonly Consumed Eggplants (Solanum mellongena depressum and Solanum gilo) in Nigeria
David Morakinyo Sanni
Department of Biochemistry, Federal University of Technology, Akure, Nigeria
To cite this article:
David Morakinyo Sanni. Partial Purification and Characterisation of Polyphenol Oxidase from Two Commonly Consumed Eggplants (Solanum mellongena depressum and Solanum gilo) in Nigeria. Biochemistry and Molecular Biology. Vol. 1, No. 1, 2016, pp. 1-10. doi: 10.11648/j.bmb.20160101.11
Received: May 31, 2016; Accepted: June 12, 2016; Published: July 6, 2016
Abstract: Polyphenol oxidase (PPO) catalyzes in the presence of oxygen, the oxidation of mono- and di- phenols to o-quinones. The browning in fruits, vegetables and their processed products occurs as a result of this oxidation reaction. PPO in this study was isolated and characterized from two species of garden egg; Solanum mellongena depressum and Solanum gilo. Extracts were purified using a combination of ammonium sulphate precipitation, ion exchange chromatography on DEAE sephadex and gel filtration on Sephadex G-200 column. The effect of pH and temperature were carried out, pH and thermal stability, kinetic and substrate specificity, the effect of inhibitors and activator on the enzyme activity was investigated. About 6% protein yield for both species and a purification fold of 30.0 and 41.0 for S. depressum and S. gilo respectively was achieved. The optima pH of activity were found to be 4.0 – 4.5 and 7.0 for S. depressum and 4.0 and 8.0 for S. gilo while 4.5 and 8.0 were obtain in presence of SDS, the activity of the PPO increased in the presence of small concentration of SDS in all the pH investigated. The temperature optimum for both species was observed at 30°C. The PPO were stable at 30°C retaining about 88% and 87% of initial activity after 60minutes for S. depressum and S. gilo respectively while PPO from S. depressum was inactivated after 40min and 80°C and 70°C. A minimal remaining activity of 5% was observed at 80°C after 60min incubation time. PPO was fairly stable at pH 6.0 – 8.0 retaining a percentage remaining activity of 40.3% to 50% for S depressum and 45.5% to 52.1% for S. gilo. The PPO exhibited a marked activity towards o-diphenol and lower activities for monophenols. Ascorbic acid, EDTA and SDS inhibited enzymatic activity while the Km values using catechol, DOPA and catechin as substrate were 0.3mM, 0.095mM and 1.09mM for S. depressum and 1.9mM, 0.414mM and 0.56mM for S. gilo. PPO from S. depressum exhibited a higher enzymatic activity compared to S. gilo while S. gilo retained a fairly more stable activity.
Keywords: Polyphenol Oxidase, Solanum spp., Enzyme, Activity
Polyphenol oxidase (PPO; 1, 2 benzenediol: oxygen oxidoreductase; EC 18.104.22.168), also known as tyrosinase, polyphenolase, phenolase, catechol oxidase, cresolase, or catecholase is widely found in nature . Polyphenol oxidase (PPO) is typically present in the majority of plant tissues and has received much attention from researchers in the field of plant physiology and food science, because of its involvement in adverse browning of plant products. Enzymatic browning occurs as a result of the oxidation by PPO of phenolic compounds to quinones and their eventual (non enzyme-catalyzed) polymerization to melanin pigments. It appears that oxidative browning reactions, proceeding in many foods of plant origin, generally cause deterioration in food quality by changing nutritional and organoleptic properties. These reactions significantly diminish consumer acceptance, storage life and value of the plant products. In addition to its general occurrence in plants, PPO is also found in seafood (crustacean) products, such as shrimp . PPO has been studied extensively for more than a century from numerous plant sources; attention is given to significant variations in physicochemical properties of the enzyme from different plant sources but comparison of the same plant of different species has not been established.
The garden eggplant is an economic flowering plant belonging to the family Solanaceae, apart from being a source of vegetable, has numerous health benefits which are essential for the overall development of human body and its acceptability cuts across religious, tribal, cultural and ethnic groups in Nigeria, they are also widely cultivated in America and Europe. They provide the body with water soluble vitamins which are rich in thiamin (Vitamin B1) required for normal growth and proper functioning of the heart and Nervous system, Niacin (Vitamin B6) needed for cellular respiration. Because of the economic and health importance of garden egg and the fact that some people eat it raw, some cooked in the form of sauce while others make salad from it, this research involves the purification and characterization of PPO from the two species of garden egg Solanum mellongena depressum and Solanum gilo and to establish and ascertain the specie with better and favourable physicochemical properties for storage.
2.1. Sample Collection and Preparation of Crude Extracts
Freshly harvested Solanum gilo (white) and Solanum mellongena depressum (green) were purchased from Oja Oba on Oba Adesida Road, Akure, Ondo State, Nigeria. About 200g of each species was homogenized with 600ml ice cold 25mM phosphate buffer (pH 6.8) containing 10mM ascorbic acid using a warring blender for 3min with a 60 seconds resting period to avoid local elevation in temperature. The mixture was filtered using four layers of cheese cloth. The filtrate obtained was centrifuged in a refrigerating centrifuge at 6,000 rpm for 30 min at 4°C. The supernatant obtained was stored in a refrigerator and used as crude extracts for further studies.
2.2. Determination of Polyphenol Oxidase Activity
PPO activity was determined according to the method described by Sanni and Adekunle,  measuring the initial rate of Quinone formation, as indicated by an increase in absorbance at 420 nm, using visible spectrophotometer. Change in absorbance was recorded after incubation at room temperature for 10 minutes. The sample cuvette contained 0.7 ml of 10mM catechol solution in 0.1 M phosphate buffer (pH 6.8) and 0.3 ml of the enzyme solution. The blank samples contain 0.7 ml of 10mM catechol and 0.3 ml of 0.1 M buffer solution. One unit of PPO activity was defined as the amount of enzyme that caused a change in absorbance of 0.001 per minute.
2.3. Partial Purification of Crude Extracts Polyphenol Oxidase
2.3.1. Ammonium Sulphate Precipitation
About 500ml of crude extract from each species was brought to 30 to 80% ammonium sulphate (NH4)2SO4 saturation by slowly dissolving solid ammonium sulphate using magnetic stirrer. The precipitate was separated by centrifugation at 6,000 rpm for 30 minutes. The precipitate was dissolved in 40ml of 0.1M phosphate buffer (pH 6.8) and dialyzed at 4°C overnight with three changes of buffer. The dialyzed samples were used as the polyphenol enzyme source in further analysis.
2.3.2. Ion Exchange on DEAE Sephadex
Dialysed enzyme solution was placed on a DEAE-Sephadex column (2.5×40 cm) and eluted with 0.01M Potassium phosphate buffer pH 6.8 and sequentially followed by 0.025M, 0.05M, 0.075M and 0.1M (flow rate: 20 ml/hr). The eluate from DEAE sephadex column was concentrated by saturated solution of Sucrose (4 M) and further purified by gel filtration.
2.3.3. Gel Filtration
Gel filtration of the enzyme was carried out on a Sephadex G-200 column (1.5× 70 cm; flow rate: 10ml/h) with 0.1M potassium phosphate buffer pH 6.8. The fractions containing the enzyme activity were pooled and used for further investigation.
2.4. Enzyme Characterisation
2.4.1. Effect of pH on Polyphenol Oxidase Activity in the Absence and Presence of SDS
Effect of pH on PPO activity in presence and absence of SDS was carried out using buffer of pH range of 4.0-9.0, 0.1 M acetate (pH 4.0-5.0), 0.1 M phosphate (pH 6.0-7.0) and 0.1 M Tris/HCl (pH 8.0-9.0). The reaction mixture in presence of SDS contains 0.69 mM of the detergent while PPO activity was assayed according to standard assay procedure.
2.4.2. Effect of pH on Polyphenol Oxidase Stability
The effect of pH on stability was determined by preparing four fold enzyme solutions with buffer solution between pH 4.0 to 9.0 at room temperature. The enzyme solutions were in catechol for 5 hours and the initial enzymatic activity was measured after mixing using a vortex mixer while the residual PPO remaining activity were determined every hour throughout the 5 hours incubation time.
2.4.3. Effect of Temperature on Polyphenol Oxidase Activity
The assay mixture containing the PPO and Catechol was incubated for 10 minutes at different temperature of 30 to 80°C at 10°C temperature interval. The absorbance reading was taken at 420 nm according to standard assay procedure.
2.4.4. Thermal Stability
Thermal stability of the purified PPOs were determined at various temperatures by incubating the enzyme solution in 0.1 M Phosphate buffer pH 6.8 for 1 hour between the temperatures range of 30 to 80°C (at intervals of 10°C). The initial enzymatic activity was obtained and residual PPO activity were determined at 10 minutes time interval for 1 hour incubation period and compared with the initial enzyme activity.
2.4.5. Substrate Specificity
Substrate specificity was determined by using eight substrates (Catechol, DOPA, Tyrosine, Catechin, Epicatechin, Gallic acid, Protocatechuic acid and ρ-coumaric acid). 5 mM of each substrate was prepared in 0.1 M phosphate buffer (pH 6.8). Enzyme activity was assayed according to standard assay procedure.
2.4.6. Effect of Inhibitors and Activator on the Enzyme Activity
Polyphenol oxidase activity was measured in the presence of ascorbic acid, EDTA and SDS at three concentrations (1, 5, 10 mM) prepared in 10 mM Catechol (pH 6.8) and the residual activity measured according to standard assay procedure.
2.4.7. Kinetic Parameters
Michaelis Menten constant (Km) and maximum reaction velocity (Vmax) were determined using three different substrates (Catechol, DOPA and Catechin). Substrate solutions were prepared in 0.1 M Phosphate buffer (pH 6.8) at different initial concentrations (1, 5, 10, 15 and 20 mM). PPO activity was assayed according to standard assay procedure. The data were processed using Lineweaver-Burk plot.
3. Results and Discussion
PPO from the two species of garden egg; S. depressumand S. gilo purified by the combination of ion-exchange chromatography on DEAE sephadex A50 and Gel filtration chromatography on sephadex G200 with a fold purification of 30 and 41 from S. depressum (table 1 and figure 1a, ) and S. gilo (table 2 and figure 2a, ) repectively and 6% purification yield in both species and a specific activity of 1254U/mg and 1161.6U/mg for S. depressum and S. gilo respectively showed the presence of Polyphenol oxidase.
|Volume (ml)||Protein conc (mg/ml)||Activity (U/ml)||Total protein (mg)||Total activity (U)||Specific activity (U/mg)||Fold||Yield (%)|
|Ammonium sulphate ppt||30||0.9||415.9||27.0||12477.0||462.1||11||21|
|DEAE Sephadex A50||20||0.4||277.5||7.5||5550.0||738.0||18||9|
|Volume (ml)||Protein conc. (mg/ml)||Activity (U/ml)||Total protein (mg)||Total activity (U)||Specific activity (U/mg)||Fold||Yield (%)|
|Ammonium sulphate ppt||30||0.7||200.7||21.3||6021.0||282.7||10||17|
|DEAE Sephadex A50||13||0.5||194.7||6.5||2531.1||389.4||14||7|
PPO from this study was observed to reveal enzymatic activity within the pH ranges: 4.0 – 8.5. This observation is within the reported pH optimum range of 4.0 – 8.0 by Yoruk and Marshall  for plants PPOs. The observed more than one optimal pH had been reported by Heimdal et al.,  for Lettuce. This may probably be due to the presence of more than one isoform of the enzyme in two species of garden egg . Oba et al.,  also observed two isoforms of PPO from banana bud extracts which exhibited variation in their PPO activities. Other plants have been reported to exhibit pH optimum at acidic region such as Cherry and strawberry , Apple and grape . However kiwifruit , and spinach  were found to revealed optimum at alkaline pH.
The result further revealed a shift in the optimum pH of the PPO from the two species of garden egg in presence of 0.69mM SDS. The activity of the enzyme increases in the presence of this low concentration of SDS at pH range of 4.0 – 9.0 with a marked activation of 66.67% and 30.17% for S. depressum and S. gilo at pH 8.5.
|pH||S. depressum (%)||S. gilo (%)|
Different authors have reported enzymatic activation by SDS. Marques et al.,  also observed alteration in general behaviour of apple PPO with changing pH in the presence of SDS, as it is inhibited at acidic pH and activated at pH above 5.0 in the presence of 3.5mM SDS. Plants such as Prunus fruits , peach , strawberry  and broad bean  PPOs also had similar report, where SDS causes a shift in the pH optimum of the enzyme from low to higher pH values. The ability of SDS to activate the enzyme alters both its enzymatic and physical characteristics and suggests that a limited conformational change due to binding of small amounts of SDS may induce or initiate the activatition of latent enzyme . Also, most enzymes lose their biological activity upon treatment with SDS because of the drastic alteration of the tertiary and quaternary structure ofprotein .
pH stability study revealed above 50% PPO activity was still retained for PPO from S. depressum and S. gilo (figure 4a and b) at pH 7 after 5 hours of incubation.
The influence of temperature on the enzyme activity of PPO for S. depressum and S. gilo are illustrated in figure 4a and b as shown below.
The optimum temperature of 30°C obtained for PPO in S. depressumand S. gilo has been reported for PPOs from apple , banana  and mango  though a higher temperature optimum of 45°C were reported for PPOs from cocoa bean  and sunflower . Other plants PPOs showed a wide range in their optimaltemperature; lettuce, 25-35°C  and grape, 25-45°C . However, PPOs from strawberry  and cucumber  have relatively high optimum temperature compared to those from other plant sources. Optimum temperature of the enzyme activity is affected by the substrate used in the assay, as reported by Sakirogluet al.,  where dog-rose extract PPO activity toward monophenols exhibits higher optimal temperature than toward di- and triphenols considered. Integrity of the delicate three-dimensional structure of the enzyme molecule is subjected to disruption and denaturation at high temperatures. These variations in temperature may also alter the solubility of oxygen, one of the substrates required for PPO to perform its catalytic activity .
The thermal inactivation study revealed a similarity in the thermal stability of PPO from S. depressum and S. gilo (figure 5a and b) with a percentage remaining activity of 88% and 87% at 30°C, 36% and 38% at 40°C, 31% and 29% at 50°C respectively.
However, at higher temperature there was complete inactivation of PPO from S. depressum after 40 and 60 minutes incubation time at 70°C and 80°C respectively while S. gilo still retained a minimal activity of 20% and 5% at 70°C and 80°C after 60 minutes. The PPO showed a better thermal stability compared to PPOs from lettuce  and cocoa bean  which are relatively heat stable. Heat treatment up to 70°C for 5 min did not affect Lettuce PPO activity while at 90°C no activity remained after 5 min . PPO in mango skin is also relatively thermostable, requiring more than 15 min at 80°C for 50% loss of activity . Thermal stability of PPO may also be influenced by nature of phenolic substrate used during determination .
The substrates specifity on PPO obtained from S. depressum and S. gilo revealed a higher activity toward o-diphenols with lower activities toward the monophenols such as tyrosine and coumaric acid.
The activity of apple PPO on tyrosine was found to be much lower than on o-diphenols  and was suggested to be typical for PPOs. Activity toward o-diphenols only was found for PPOs from grape , field bean seed  strawberry  and sunflower seed . Lee et al.,  observed that substrate specificity of PPO is also dependent on species and cultivars. For example, in the case of DeChaunac grape PPO, caffeic acid is oxidized at much faster rates than other structurally related substances. Oba et al.,  further observed that PPO isoforms in a tissue of interest may also exhibit differential substrate specificities and variations in their relative activities toward monophenols and o-diphenols.
|Relative activity (S. depressum)%||Relative activity (S. gilo)%|
**Catechol was used as reference substrate.
|S. depressum||S. gilo||S. depressum||S. gilo||S. depressum||S. gilo|
The PPO from S. depressum and S. gilo were found to be inhibited by Ascorbic acid, EDTA and SDS as seen in table 5 above. Ascorbic acid is commonly used as an antibrowning agent in the manufacturing of fruit juices, purees, frozen sliced fruits, and canned fruits and vegetables . López-Nicolás et al.,  found ascorbic acid in combination with citric acid to be a more effective inhibitor than ascorbic acid alone. This increased inhibition is likely due to the increased stability of ascorbic acid in an acidic environment as well as inhibition of the acidic environment on the catalytic activity of the enzyme. EDTA however is a chelating agent. The active site of PPO is comprised of two copper atoms through which the enzyme interacts with its phenolic substrates and oxygen. Chelating agents have the ability to react with metals through chelation, making copper at the active site unavailable, thereby inhibiting PPO. Also, resistance of PPO to SDS as observed in this study is perhaps due to presence of disulfide bonds strengthening PPO structure . Degree of SDS activation varies greatly with plant materials. For example, PPO activity increased about 65 to 119-fold in broad bean , 19-fold in grape , 25-fold in peach , 10-fold in banana , 4-fold in potato leaf  and 7-fold in mango  by SDS under the defined experimental conditions. In general, these compounds diminish or inhibit the browning reaction rate by means of eliminating from the reaction an active reaction element(s). PPO from different sources may react similarly with inhibitor compounds. However, effectiveness of inhibitors against different PPOs could significantly vary and, therefore, specific control measures for individual systems would also be needed .
Reaction rate at different catechol, DOPA and catechin concentrations showed that polyphenol oxidase from S. depressum and S. gilo obeys Michealis-Menten kinetics. Table 6 shows summary of kinetic parameters.
|S. depressum||Km (mM)||0.3||0.095||1.09|
|S. gilo||Km (mM)||1.9||0.414||0.56|
Km values 0.3mM, 0.095mM and 1.09mM for S. depressum and 1.9mM, 0.414mM and 0.56mM for S. gilo from substrates respectively obtained using Lineweaver-Burk plot, which in comparison with the other PPO from different fruitshad lower affinity. Marques et al.,  observed that Km value for apple was 34.0mM catechol and 3.1mM 4-Methylcatechol while Kmvalues for Grape are 3.2mM Chlorogenic acid 4.3mM Catechin . Plant PPOs are capable of oxidizing a wide range of o-diphenols, the primary substrates with varied oxidation rates and affinities of different orders of magnitude and the native concentrations of natural phenols vary from plant to plant. The variability of Vmax values amongst the species was related to the absolute concentration of PPO in their respective extracts, since absolute concentration of enzyme (E) shows direct proportionality with maximum enzyme activity (Vmax). This result conforms to previous reports stated which noted significant differences and marked variations in PPO kinetic parameters between enzyme extract of different species of plants and their substrates.
This study revealed the activity of PPO in S. depressum and S. gilo and its physicochemical properties. The enzyme was active in all the pH range between 4.0 – 8.5 for both species with the same optimum temperature of 30°C. The PPOs exhibited similar thermal characteristic with a remaining activity of 88% and 87% at 30°C, 36% and 38% at 40°C, 31% and 29% at 50°C for S. depressum and S. gilo respectively after a period of an hour. Higher activity was observed towards o-diphenols and lower enzyme activity towards monophenols, while EDTA, ascorbic acid and SDS showed varying level of PPO inhibition from S. depressum and S. gilo.