Selected Properties of Polyphenol Oxidase Obtained from Ispir Sugar Bean

Abstract

Polyphenol oxidase enzyme was isolated from Ispir sugar bean by ammonium sulphate precipitation and its biochemical properties were investigated. For this purpose, K_M and V_max values for optimum conditions of pH, temperature, and ionic strength were determined for catechol, catechin, and chlorogenic acid as substrates. Enzyme activities were measured spectrophotometrically at 420 nm using the same substrates at optimum conditions. K_M values were found to be 2.4875, 1.3154, and 2.2487 M for catechol, catechin, and chlorogenic acid, respectively. V_max values were 3.1480, 0.6130, and 0.5039 EU/ml.min for the same substrates, respectively. These results indicated that catechol was used as a subsrate for inhibition studies. For catechol substrate, dithiothreitol, glutathione, thiourea, and L-cysteine chlorid were inhibitors. For these inhibitors, K_i constants were calculated from Lineweaver-Burk plots and inhibiton types were estimated. Moreover, I₅₀ values were also determined. The most effective inhibitor was found to be glutathione.

Keywords:

• Polyphenol oxidase
• Ispir sugar bean
• Glutathione
• Catechol
• Inhibition

INTRODUCTION

Polyphenol oxidase (PPO) is a major enzyme responsible for the browning reaction in damaged plant tissues and fruits.[1 2] Enzymatic browning in plants occurs by the oxidation of phenolic compounds, which are oxidized to quinones in the presence of O₂ and PPO following the polymerization of quinones.[3 4] In some food products, browning reactions generally result in a good appearance in terms of colour, but they lead to undesirable results with respect to texture, sweetness, and overall flavour. Therefore, inhibition studies have gained more importance for these types of reactions in food and vegetable processing technology.[5] In this regard, PPO has gained more attention in food technology. The agricultural products are also harmed by enzymatic browning reactions during harvesting, storage, and processing. These problems cause low-quality products and economic losses.[1 6 7] Enzymatic browning, catalyzed by PPO, occurs when plant tissues are damaged. This leads to an economic problem for producers, processors, and consumers. The main step in enzymatic browning is the oxidation of phenolic compounds to corresponding quinones by PPO in the presence of oxygen. Then, the quinones condense to form darkened pigments.[8] These reactions, known as enzymatic browning, are not generally desirable for the food industry but can be used for preparation of dark tea. However, the PPO values, especially of the Ispir sugar bean, gain a special interest for study because beans are an important source of nutrients.

Sugar bean grown in many parts of the world is the most important economic species of the genus Phaseolus. The main domestication areas of dry beans are Central and South America, Eastern and Southern Africa, and East Asia.[9 10] It is widely cultivated as an agricultural crop throughout the world both for dry beans and fresh pods. The world leaders in production of dry beans are Brazil, India, and China with 3.17, 3.00, and 1.23 million tonnes, respectively. The total dry bean production in the world and Turkey is 19.70 and 0.20 million tonnes, respectively. In recent years, the dry bean cultivars of ‘Ispir’ have increasingly become more popular, not only in Erzurum, but also in Turkey as a whole because of their high quality seeds.[10 11]

On the other hand, people need to consume a variety of plant foods to obtain all the amino acids necessary for their body to form complete proteins. There have been many researchs on finding a way to optimize their intake of legumes, such as dry bean, chickpea, lentil, and pea, as part of a healthier lifestyle and to make their body stronger by being a part of an alternative treatment against illnesses. Dry bean is a staple food all over the world and one of the best sources of soluble fiber. It is low in fat and high in good quality protein. The soluble fiber in beans helps to lower the level of damaging LDL cholesterol in the blood and so lower the heart-disease risk. Additionally, it fends off unwanted peaks and valleys in blood glucose levels, especially valuable to people with diabetes by slowing down carbohydrate absorption.[12] Dry bean is also highly rich in starch, protein, and dietary fiber. It is also an excellent source of iron, copper, magnesium, potassium, selenium, molybdenum, thiamine, vitamin B6, and folic acid. Furthermore, dry bean is a base meal of the populations indigenous to Central and South America; Central, Eastern, and Southern Africa; and East Asia where it is variously consumed.[13]

There are many works related to PPO from different plants, such as wheat,[14] Allium sp.,[15] wheat flour,[16] Beta vulgaris L.,[17] tea leaf,[18] lettuce,[19] dog-rose,[20] Ferula sp.,[21] peaches,[22] apples,[23 24] grapes,[25 26] rosmarinus officinalis L.,[27] Salvia species,[28] Thymbra spicata L. var. spicata,[29] green bean,[30] dill,[31] and Thymus.[32] In this work, polyphenol oxidase extracted from Ispir sugar bean was characterized to determine the substrate specificity, kinetic parameters, pH stability, inhibition effects of various inhibitors, optimum pH, and temperature.

MATERIALS AND METHODS

Plant Material

Ispir sugar bean has been used as research material in this study. Ispir sugar bean was collected in the spring sessionfrom a field near Ispir, which is a town located in the northeast of Erzurum, Turkey. The sugar bean samples were then stored at 4°C until used in the study.

Extraction and Partial Purification of PPO

Five grams of each Ispir sugar bean sample was frozen in liquid nitrogen and then powdered. The powdered material was added to 100.0 mL of extraction buffer, 0.5 M phosphate buffer, pH 7.3, containing 0.5% polyethylene glycol and 10 mM ascorbic acid. The crude extract samples were centrifuged at 48,000 g for 30 min at 5°C. The supernatant was brought to 80% (NH₄)₂SO₄ saturation with solid (NH₄)₂SO₄. The precipitated PPO was separated by centrifugation at 48,000 g for 30 min. The precipitate was dissolved in a small amount of 5 mM phosphate buffer (pH 5.2) and dialyzed at 4°C in the same buffer for 24 h with four changes of the buffer during dialysis. The dialyzed sample was kept at 4°C and then was used as the PPO enzyme source in the following experiments.

Determination of PPO Activity

PPO activity was determined by measuring the increase in absorbance at 420 nm with a Beckman spectrophotometer (DU 730; Beckman Coulter Inc., Fullerton, CA, USA). The sample cuvette contained 0.10 mL of the enzyme solution and 2.90 mL of substrate solution in various concentrations. The blank sample contained only 3.00 mL of substrate solution. The reaction was carried out at various temperatures and pH values with the substrates mentioned as follows. PPO activity was calculated from the linear portion of the curve.[33] One unit of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001/min.

Characterization of PPO

Effect of pH

PPO activity was determined with three different substrates (catechol, catechin, and chlorogenic acid) at a concentration of 10 mM. Appropriate buffers (0.1 M citrate/0.2 M phosphate for pH 4.0–5.5, 0.2 M phosphate for 5.5–7.0, and Tris–HCl for 7.0–10.0) were used for the determination of optimum pH for PPO. The optimum pH values obtained from this assay were used in all subsequent experiments. PPO activities were measured in 0.1 M phosphate buffer (pH 5.0–7.0) and 0.1 M Tris–HCl buffer (pH 7.0–8.5) to determine the stability of PPO. The activity measurements were performed every 2 days by using catechol as a substrate under optimum conditions.

Effect of temperature

For determining optimum temperature values of the enzyme, PPO activity was measured at different temperatures in the range 5–85°C using the three different substrates as indicated above. The desired temperatures were provided by using an ice bath for temperatures under 20°C and a constant-temperature circulator for temperatures above 20°C.

Effect of ionic strength

Ionic strength effect on the enzyme was studied at 10.0 mM substrate concentration using different concentrations of buffers.

Enzyme kinetics and substrate specificity

For the determination of Michaelis constant (K _M) and maximum velocity (V _max) values of the enzyme, PPO activities were measured with three different substrates varying concentrations in the range of 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, and 15.0 mM under optimized conditions of pH, ionic strength, and temperature. K _M and V _max values of PPO for each substrate, were calculated from a plot of 1/V versus 1/[S] by the method of Lineweaver and Burk.[34] Substrate specificity (V _max/K _m) was calculated by using the data obtained on the Lineweaver-Burk plot.

Effect of inhibitors

Inhibitor effects on PPO activity were studied by using the following inhibitors: dithiothreitol, glutathione, thiourea, and L-cysteine chlorid at five different concentrations of inhibitors with 10.0 mM catechol as a substrate at pH 5.5. Percent activity graphs were drawn from these results to find both I₅₀ values. Next, PPO activities were measured at three constant inhibitor concentrations with the inhibitors indicated above using five different concentrations of the substrate. 1/V and 1/[S] values obtained from these activity measurements were used for drawing Lineweaver-Burk graphs. Finally, K _i constant values were calculated from the graphs.

RESULTS AND DISCUSSION

Extraction and Purification of PPO Enzyme

PPO activity of the precipitate of 0–80% (NH₄)₂SO₄ saturation was found to be the highest, and this saturation point was used for all the extraction processes. Polyethylene glycol was used during extractions to bind the phenols that could inactivate the PPO. It is well documented that oxidation of phenolics by PPO produces quinones that would inhibit PPO.[35] Therefore, ascorbic acid was also used to reduce quinones to phenolic substrates during extraction.

Characterization of Ispir Sugar Bean PPO

The enzyme samples obtained by ammonium sulfate precipitation were used for the characterization of the PPO. Next, the enzyme samples were dialyzed and kept at 4°C. These samples were used as the PPO enzyme source for the following experiments.

Effect of pH

Activity of purified enzyme was measured with three different substrates to determine optimum pH for each substrate (Table 1). Optimum pH with catechol substrate was found to be phosphate buffer pH 5.5, Tris-HCl buffer pH 8.0 with (+) catechin, and phosphate buffer pH 7.0 with chlorogenic acid. Most of the plants, vegetables, and fruits generally show maximum activity at or near neutral pH values.[36] Different optimum pH values for PPO obtained from various sources are reported in the literature. For example, it is reported that optimum pH values are 5.5 for strawberry;[37] 6.0 for DeChaunac grape;[38] 7.0 for Amasya apple,[24] aubergine,[39] Yali pear,[40] and Ferula sp.;[21] 7.5 for Allium sp.;[15] and 8.5 for dog-rose[20] using catechol as a substrate. However, when using 4-methylcatechol as a substrate the pH optimum is 4.5 for strawberry, 6.0 for aubergine,[39] 8.5 for dog-rose,[20] and 9.0 for Amasya apple,[24] and 8.5 for dog-rose[20] using dopamine as a substrate and 7.0 for dog-rose[20] and 8.6 for Amasya apple[24] using pyrogallol as a substrate. PPO activity varies with the source of enzyme and substrate within a relatively wide range of pH. Although, in most cases, pH optima have been reported between 4.0 and 7.0. It is noteworthy that the optimum pH can also be affected by the type of buffer and the purity of enzyme.[39]

Table 1 Optimum pH and temperature, and K _M and V _max and V _max/K _M values of Ispir sugar bean's PPO

Substrate	Optimum pH	Optimum temperature (°C)	K M (M)	V max (EU/mL.min)	V max/K M (EU/mmol.s)
Catechol	5.5	40	2.4875	3.1480	0.021
Catechin	8	50	1.3154	0.6130	0.008
Chlorogenic acid	7	20	2.2487	0.5039	0.004

The stability of the enzyme at different pH conditions (4.0–8.5) over a period of 10 days was investigated using catechol as substrate. It was found that PPO activity of Ispir sugar bean was highest at pH 6.0 but decreased at similar rates at each of the pH values studied (Fig. 1).

Figure 1 Stability pH of Ispir sugar bean's PPO at various pHs for catechol substrate.

Effect of temperature

The temperature effects on PPO activity of Ispir sugar bean were studied in the range of 5 and 85°C with each of the three substrates used in the experiments (Table 1). As seen in the table, all optimum temperatures are substrate dependent. The optimum temperature is 40°C for catechol, 50°C for catechin, and 20°C for chlorogenic acid. It is reported that the most efficient temperature for PPO is 15°C for Amasya apple,[24] 20°C for DeChaunac grape,[38] 25°C for dog-rose,[20] 30°C for aubergine,[39] 12°C for Ferula sp.,[21] and 40°C for Chinese cabbage[41] using catechol as a substrate; 20°C for dog-rose,[20] 30°C for aubergine,[39] 25°C for Ferula sp.,[21] and 56°C for Amasya apple[24] using 4-methylcatechol as a substrate; and 15°C for dog-rose[20] and 70°C for Amasya apple[24] using pyrogallol as a substrate.

Enzyme kinetics and substrate specificity

In this study, three widely used substrates (catechol, catechin, and chlorogenic acid) were selected for kinetics studies. The K _M and V _max values were calculated from substrate's Lineweaver-Burk graphs and are shown in Table 1. As seen in Table 1, the PPO of Ispir sugar bean has a great affinity towards catechin (K _M = 1.3154 M) of the three subtrates and the catechol derivatives tested highest, as seen from the high V _max/K _M ratio (0.021 EU/mmol.s). When the V _max values for the three substrates were compared, it was found that the V _max for catechol was higher than for the other substrates. Consequently, catechol was used as a substrate in other kinetics studies.[42] This observation was similar to that of the work on PPO from dog-rose,[20] ferula sp.,[21] and Amasya apple.[24] There are a number of compounds, such as dopamine,[20 41] catechol,[20 24 37] chlorogenic acid, L-dopa,[20 24 37 41] pyrogallol,[20 24 37 41] caffeic acid,[37 38] p-cresol,[20 37 41] tyrosine,[20 38] and 4-methylcatechol,[20 24 37] which were used as substrates for polyphenol oxidase in the literature.

Effect of inhibitors

I₅₀ values are shown in Table 2 for each inhibitor. K _i values and inhibition modes for four inhibitors are given in Table 3. It was concluded from the Lineweaver-Burk plots that the inhibition modes for all four inhibitors are noncompetitive. The strongest inhibitor was found to be glutathione followed by dithiothreitol, thiourea, and L-cysteine chlorid, respectively. There are a number of inhibitors, such as sodium metabisulphite,[20 38] ascorbic acid,[20 38] glutathione,[27 38] sodium diethyldithiocarbamate,[38] L-cysteine, sodium azide, tannic acid, benzoic acid, and β-mercaptoethanol,[20 27] that were used by researchers to prevent enzymatic browning. L-Cysteine can easily form complexes with quinones and, thereby, inhibiting secondary oxidation and polymerisation reactions.[43] L-Cysteine can also act as a reducing agent.[37] Ascorbic acid reduces quinones to hydroquinones but does not directly inhibit PPO.[44] It will prevent enzymatic browning only as long as it is present in the reduced form. This prevents the formation of key intermediates and inhibits the activity of the oxidase.[45]

Table 2 I₅₀ values for four inhibitors for PPO

Inhibitors	I50 (M)
Dithiothreitol	7.41 × 10^−6
Glutathione	7.66 × 10^−6
Thiourea	6.30 × 10^−6
L-Cysteine chlorid	6.83 × 10^−6

Table 3 K _i values and inhibition modes for four inhibitors for PPO

Inhibitors	[I] (M)	K i (M)	Average values of K i (M)	Type of inhibition
Dithiothreitol	4.0 × 10^−6	9.97 × 10^−7	1.18 × 10^−6	Noncompetitive
	8.0 × 10^−6	1.03 × 10^−6
	1.6 × 10^−5	1.52 × 10^−6
Glutathione	1.4 × 10^−6	7.07 × 10^−7	7.04 × 10^−7	Noncompetitive
	2.3 × 10^−6	6.20 × 10^−7
	4.7 × 10^−6	7.87 × 10^−7
Thiourea	1.8 × 10^−6	1.05 × 10^−6	1.23 × 10^−6	Noncompetitive
	3.6 × 10^−6	1.02 × 10^−6
	9.6 × 10^−6	1.63 × 10^−6
L-Cysteine chlorid	1.7 × 10^−6	1.28 × 10^−6	1.41 × 10^−6	Noncompetitive
	3.3 × 10^−6	1.19 × 10^−6
	6.5 × 10^−6	1.77 × 10^−6

CONCLUSION

The charecterization of PPO is important because of the browing reactions, which occur in fruits and vegetables. In this respect, PPO enzyme of Ispir sugar bean was partially purified and PPO activity was measured spectrophotometrically using selected substrates and inhibitors. The best substrate of PPO enzyme was found to be catechol (high V _max/K _M ratio) while glutathione is the most effective inhibitor. The four inhibitors used in this study caused inhibition effects at a concentration of 10⁻⁶ M and can be used safely in the protection of sugar bean.