Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 211–217

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Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits Karla A. Batista ∗ , Gustavo L.A. Batista, Guilherme L. Alves, Kátia F. Fernandes Laboratório de Química de Proteínas, Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas – ICB II, Universidade Federal de Goiás, PB 131 Goiânia, GO, Brazil

a r t i c l e

i n f o

Article history: Received 4 October 2013 Received in revised form 22 February 2014 Accepted 23 February 2014 Available online 2 March 2014 Keywords: Solanum lycocarpum Polyphenol oxidase Thermal inactivation Kinetic parameters Inhibitors

a b s t r a c t In this work a polyphenol oxidase (PPO) from Solanum lycocarpum ripe and unripe fruits was studied. The unripe fruits presented higher activity than ripe for both fresh fruits and dried pulp flours. The purification procedure was based on freezing precipitation and a sixfold purification factor was obtained. The SDSPAGE of the partially purified PPO showed two bands around 47 and 68 kDa. Optimal conditions for enzymatic studies were determined to be pH 6.0–6.5 and 28 ◦ C. The partially purified PPO presented high activity toward catechol (Vmax 3.42 U mL−1 and Km 6.47 mM) and 4-methylcatechol (Vmax 3.01 U mL−1 and Km 0.15 mM) and low activity toward phenol being classified as a catecholase type polyphenol oxidase. S. lycocarpum PPO was sensitive to inhibitors such as l-cysteine, sodium metabisulfite, ascorbic acid, thiourea and citric acid. l-Cysteine was the most effective inhibitor, presenting a competitive inhibition. The results from kinetic and thermodynamic parameters for the thermal inactivation evidenced that the partially purified PPO is a biocatalyst, whose inactivation process is related to aggregation of partially unfolded enzyme molecules. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polyphenol oxidase (PPO) is a common copper-containing enzyme, which catalyzes two distinct reactions involving molecular oxygen: the o-hydroxylation of monophenols to o-diphenols (cresolase activity; E.C. 1.14.18.1) and the oxidation of o-diphenols to o-quinones (catecholase activity; E.C. 1.10.3.2) [1–3]. This enzyme is important because it is responsible for skin, eye, inner ear and hair melanization, and browning in fruits and vegetables [4]. PPO can be found in many living organisms such as fungi, mammals, birds, insects and a diversity of plants. In plants, it can be found free in the cytosol or associated with the thylakoid membrane of chloroplasts [5]. Solanum lycocarpum is a common and abundant plant in the Brazilian cerrado [6], whose fruits are an abundant and inexpensive source of PPO [7]. In addition, the high productivity of S. lycocarpum fruits contributes to the use of this vegetal as promising source of PPO. Although the activity of PPO is undesirable during processing of different foods, there are many technological processes based on the reaction of this enzyme.

∗ Corresponding author. Tel.: +55 62 3521 1492; fax: +55 62 3521 1190. E-mail address: [email protected] (K.A. Batista). http://dx.doi.org/10.1016/j.molcatb.2014.02.017 1381-1177/© 2014 Elsevier B.V. All rights reserved.

In the recent years, the use of PPO in industry, cosmetics, clinical and analytical applications has received much attention from the researchers. In food industry, the use of PPO was found to be valuable for removal of the astringent and bitter taste of cocoa beans and enzymatic cross-linking of proteins as an alternative to the use of transglutaminase [8]. Out of food industry, PPO have been used for commercial production of L-DOPA [9], for the removal of phenolic compounds from wastewater [10], as biosensor to detect pollutants in environmental samples [11], or for the analysis of thiol-containing compounds [12]. In spite of its ubiquitous distribution in nature, the majority of the PPO sources present low productivity and the purification of this enzyme is frequently obtained through multi-step procedures. Moreover, in general polyphenol oxidases present low thermal stability restricting their industrial use. Each potential use of PPO demands specific characteristics. In this sense, the aim of this work was to purify and characterize the PPO extracted from S. lycocarpum. The substrate specificity, kinetic parameters and degree of inhibition by general PPO inhibitors were studied in order to predict the behavior of the partially purified PPO from S. lycocarpum. The characterization of thermal behavior of partially purified PPO was also determined in terms of denaturation kinetic and thermodynamic parameters.

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2. Materials and methods 2.1. General experimental procedures PPO substrates (catechol, 4-methylcatechol, pyrogallol, phenol) and inhibitors (ascorbic acid, citric acid, thiourea, sodium metabisulfite, l-cystein) were acquired from Sigma–Aldrich. The electrophoresis reagents (acrylamide, N,N methylenebis(acrylamide), TEMED, molecular markers) were acquired from GE Healthcare Life Sciences. All other reagents were of analytical grade.

method described by Laemmli [15]. Electrophoresis was run at 25–40 mA for 4 h at room temperature. Gels were stained according to the methodology described by Blum et al. [16], using silver nitrate. Considering the high hydrophobicity of S. lycocarpum PPO, the samples containing the partially purified enzyme (20 ␮L containing 40 ␮g protein) were additionally treated with 5% Triton X100 solution in a ratio of 1:10 (protein solution:triton) and heated for 5 min at 100 ◦ C in a bath water. This agent binds to protein in proportion to their hydrophobicity and is useful in the study of proteins that differ slightly in their hydrophobic character and that are not separated by ordinary SDS gel electrophoresis [17].

2.2. Plant material 2.7. pH and temperature profile of PPO Unripe and ripe fruits of S. lycocarpum used in this study were collected at Fazenda Estrela, Silvânia, GO, Brazil. The PPO activity tests were conducted using fresh material and flour produced by milling the dried pulp. The pulp of decorticated S. lycocarpum fruit was dried at 40 ◦ C in an air-forced oven until constant weight. Dry samples were milled, sieved at 425 mm and stored in air-tight vials at 4 ◦ C until use. 2.3. Enzyme extraction The crude extract was obtained according to methodology described by Carneiro et al. [13], using polyvinylpyrrolidone (PVP) as phenol scavenger. Briefly: 20 g of fresh fruit pulp was mixed with 100 mL of 50 mmol L−1 sodium phosphate buffer pH 6.5, supplemented with 1% (w/v) PVP. This mixture was crushed in a blender for 1 min, vacuum filtered and the extract used to determine the PPO activity. The crude extract of the fruit flour was obtained adding 10 g of the flour in 100 ml of 50 mmol L−1 sodium phosphate buffer pH 6.5, supplemented with 1% PVP (w/v) under stirring for 30 min at 4 ◦ C. The material was vacuum filtered and the extract used to determine the PPO activity.

The optimum pH of the partially purified PPO was investigated by measuring its activity at room temperature (28 ◦ C) at pH ranging from 3 to 9. The tests were carried out using 50 mmol L−1 sodium acetate buffer for pH ranging from 3 to 5; 50 mmol L−1 sodium phosphate buffer in the test at pH 6, 6.5 and 7, and 50 mmol L−1 Tris buffer for tests at pH 8 and 9. The effect of temperature on PPO activity was carried out by assaying the reaction mixture at various temperatures ranging from 10 to 70 ◦ C. 2.8. Thermal stability and inactivation kinetics of partially purified PPO The thermal stability of S. lycocarpum PPO was evaluated by measuring the remaining activity of pre-incubated enzyme at different temperatures (30–65 ◦ C) in sodium phosphate buffer solution (50 mmol L−1 , pH 6.5) for 2 h. The pre-incubated enzyme was then cooled in ice bath before the enzyme assay (Section 2.3). The percentage of remaining activity after each treatment was calculated according to the following equation: Remaining activity (%) =

2.4. PPO activity assay PPO activity was measured using the method described by Carneiro et al. [13], using sodium phosphate buffer. The enzyme extract (100 ␮L) was added to 2.9 mL of 46 mmol L−1 catechol solution in 50 mmol L−1 sodium phosphate buffer (pH 6.5) to initiate the reaction. The reaction was left to proceed for 1 min at room temperature and the absorbance was measured at 420 nm. One unit of PPO (U) was defined as the amount of PPO that produces an increase of 0.1 in absorbance per minute of reaction. PPO specific activity was determined from ratio between enzyme activity (U mL−1 ) and protein content (mg mL−1 ). The protein content was determined using the Bradford [14] protein dye-binding method with bovine serum albumin as a standard.

Where Ai is the enzyme activity after incubation, and Ao is the enzyme activity before incubation.The thermal denaturation kinetic of PPO was described as first order [18,19]. In this sense, for constant extrinsic/intrinsic factors, the kinetic of a first-order reaction can be described by the following equation: At = e−kd t A0 where A0 is the initial enzyme activity, At is the residual activity at time t, t is the treatment time (h), and kd is the inactivation rate constant at the temperature studied. The half-life (t1/2 ) value for PPO thermal denaturation was calculated as:

2.5. Partial purification t1/2 = Crude PPO extracts were partially purified by freezing. 100 mL of PPO extract were incubated at −18 ◦ C for 18 h. Subsequently, the material was defrosted and centrifuged at 5000 × g for 10 min at 4 ◦ C. The precipitate was homogenized in 1 mL of 50 mmol L−1 sodium phosphate buffer pH 6.5, containing 0.1% SDS (w/v). The degree of purification was determined by measuring specific activity before and after precipitation. 2.6. Electrophoresis (SDS-PAGE) The partially purified PPO was analyzed using 10% denaturing polyacrylamide gel electrophoresis (SDS-PAGE) according to the

Ai × 100 A0

ln(2) kd

The D-value is the time (min) necessary to reduce the initial activity 90%. It was related to kd -values and mathematically expressed by: D=

ln(10) kd

In general, Arrhenius’ law is used to describe the temperature dependence of kd -values, and it is algebraically given by: ln kd = ln k0 −

Ea R·T

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 211–217

where k0 is the Arrhenius constant, kd is the inactivation rate constant at the temperature studied, Ea is the activation energy, R is the universal gas constant (8.3145 J mol−1 K−1 ) and T is the absolute temperature (K). The change in Ea can be obtained from the slope of the Arrhenius plot (regression of logarithm of reaction rate constants (ln kd ) versus reciprocal of absolute temperature), as described below: Ea slope = R The values of the activation energy (Ea ) and inactivation rate constant (kd ) allowed for the determination of different thermodynamic parameters, such as variations in enthalpy, entropy, and Gibbs free energy. Change in the enthalpy (H) for each temperature can be calculated according to: H = Ed − RT The values of variation in free energy of inactivation (G) at different temperatures were calculated from the first-order rate constant of the inactivation process by: G = −RT ln

k h d

KT

where h is the Planck constant (6.6262 × 10−34 J s) and K is the Boltzmann constant (1.3806 × 10−23 J K−1 ). Using the equations above, the activation entropy (S) for PPO can be calculated as: S =

H − G T

213

Table 1 PPO activity (U g−1 dry weight) determined in the extracts of fresh fruits and dried pulp flour. Crude extract

PPO activity

Fresh fruit

Unripe Ripe

214.91a ± 24.91 45.45b ± 4.63

Dried pulp flour

Unripe Ripe

171.95a ± 30.23 12.26c ± 1.14

Results are presented as the mean ± standard deviation of three determinations. The values followed by the same letter are not statistically different (p > 0.05).

where A indicates the variation of absorbance in the absence of the inhibitor and Ai indicates the variation of absorbance in the presence of the inhibitor. In order to determine the inhibition constant (Ki ) for each studied inhibitor, assays were conducted measuring the PPO activity against different concentrations of catechol (10–50 mmol L−1 ) at a constant inhibitor concentration. Values 1/V and 1/S were employed to draw Lineweaver–Burk graphs and the Ki values were obtained using the following equation:



1 [I] Vmax · 1+ = V Km Ki

 1 ·

[S]

+

1 Vmax



1+

[I] Kii



where V is the initial reaction rate, [S] is the initial substrate concentration, Vmax is the maximum reaction rate attained at infinite initial substrate concentration, Km is the Michaelis–Menten constant, Ki is the inhibition constant for the enzyme-inhibitor complex and Kii is the inhibition constant for the enzyme–substrate–inhibitor complex.

2.9. Substrate specificity and kinetic parameters 2.11. Statistical analysis The substrate specificity of the PPO was determined by measuring its activity against catechol [13], 4-methylcatechol [20], pyrogallol [21], and phenol [22]. Michaelis–Menten constant (Km ) and maximum reaction velocity (Vmax ) were determined for three substrates: catechol (5–50 mmol L−1 ), 4-methylcatechol (0.1–1 mmol L−1 ) and pyrogallol (1–15 mmol L−1 ). Data from linear range of Michaelis–Menten plot were used to construct a double-reciprocal plot according to the method described by Lineweaver and Burk [23]. The values of Vmax can be deduced from the reciprocal of the intercept of the straight line on the ordinate and the values of Km from the negative reciprocal of the intercept on the abscissa, using the following equation: 1 1 Km + = V Vmax Vmax · [S] where V is the initial reaction rate, [S] is the initial substrate concentration, Vmax is the maximum reaction rate attained at infinite initial substrate concentration and Km is the Michaelis–Menten constant [24]. 2.10. Inhibition studies The effects of the inhibitors ascorbic acid, citric acid, thiourea, sodium metabisulfite and l-cysteine on S. lycocarpum PPO activity were studied. To determine the effects of the inhibitors, reaction containing 46 mmol L−1 catechol and a constant amount of enzyme (3.2 U) in 100 mmol L−1 phosphate buffer (pH 6.5), were run at room temperature (28 ◦ C), in the presence and absence of the inhibitor. The percentage of inhibition was calculated according to the following equation: %inhibition =

A − Ai × 100 A

All tests were conducted according to a completely randomized model. Statistica software (Statistica 6.0, Statsoft Inc., Tulsa, USA, 1997) was used to perform analysis of variance (ANOVA) followed by the Tukey test to determine the significant differences among the means. The level of significance used was 95%. 3. Results and discussion 3.1. PPO activity The results for PPO activity extracted from S. lycocarpum fruits are shown in Table 1. As can be observed the unripe fruits presented higher activity (p < 0.05) than ripe fruits for both fresh fruits and dried pulp flour. The effect of maturation on the PPO activity has been reported by previous researches [25–28]. According to Oliveira-Júnior et al. [7], the PPO activity decreases following the fruit ripening. In this study, the PPO activity of fresh unripe fruits was about fivefold higher than those observed in ripe fruits. In addition, the dried pulp flour of unripe fruits presented an activity 14-fold higher than dried pulp flour of ripe S. lycocarpum fruits. Although the dried pulp flour of ripe fruits presented a 73% decreasing in the PPO activity compared to fresh ripe fruits, the drying process was not associated with a decreased PPO activity in the unripe fruits. The reduction in the PPO activity observed in the dried pulp flour of the ripe fruits may be due to the physiological changes occurring during the ripeness. During the fruit ripeness the thylakoids membranes are destroyed. Consequently, the membrane-associated components such as enzymes and proteins are metabolized and the polyphenol oxidase probably is directed to the intracellular turnover of proteins [29]. Therefore, based on these results, the further purification procedures were conducted using unripe fruits from S. lycocarpum.

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Table 2 Purification of polyphenol oxidase from Solanum lycocarpum fruits. Fresh fruit

PPO activity (U mL−1 ) Protein content (mg mL−1 ) Specific activity (UE mg−1 protein) Purification factor

Dried pulp flour

Crude extract

Freezing precipitated

Crude extract

Freezing precipitated

14.70b ± 1.70 2.47b ± 0.25 5.95b ± 0.69

13.20b ± 2.69 2.06c ± 0.04 6.41b ± 1.30 1.08

14.63b ± 2.57 2.96a ± 0.06 4.94c ± 0.87

23.07a ± 1.91 1.11d ± 0.05 29.78a ± 1.72 6.03

Results are presented as the mean ± standard deviation of three determinations. Within lines, values followed by the same letter had no significant difference (p > 0.05).

3.2. Effect of partial purification on the PPO activity The results of enzyme activity, protein content, specific activity and purification factor obtained in the PPO purification procedure are shown in Table 2. As can be seen, there was no statistical difference (p > 0.05) between the PPO activity in the crude extract and the freezing precipitated fraction obtained from fresh fruits. However, the PPO activity observed in the freezing precipitated fraction from flour was 57.7% higher than that obtained in the crude extracts (fresh fruit and dried pulp flour) and 74.8% higher than the freezing precipitated fraction of fresh fruits. The production of flour using drying process followed by fine milling, likely resulted in a more efficient cellular disruption. Consequently, a better extraction of cytosolic and thylakoid membrane associated PPO was favored. The freezing process and the ordering in the water structure favored protein aggregation and their precipitation. This process is greatly enhanced for thylakoid membrane bound proteins, due to the large hydrophobic area on the surface of these proteins. The SDS treatment used to solubilize the precipitated material releases these proteins allowing the detection of their activity. The extract obtained by crushing the pulp with the blender was less effective in cellular disruption and likely extracted mainly cytosolic proteins. Regarding to the protein content in the crude extracts, the efficiency of the extraction was enhanced 19.8% using the flour instead of fresh fruit. This result reinforces the hypothesis that drying and milling associated processes enhanced the cell wall rupture and favored the release of the cellular content. Nevertheless, the protein content in the precipitated fraction from the S. lycocarpum dried pulp flour was 62.5% lower than the crude extract. The simultaneous release of cellular compounds able to associate with proteins forming insoluble high molecular aggregates may be the explanation for this finding. The lower content of protein in the precipitate fraction of S. lycocarpum dried pulp flour lead to an increase of the specific activity, indicating that freezing was an effective methodology for partial purification of PPO from S. lycocarpum fruits. As showed in Table 2, the freezing of the crude extract from S. lycocarpum flour resulted in a sixfold purification, in a single step procedure. The PPO partial purification was confirmed by denaturizing polyacrylamide gel electrophoresis (Fig. 1). The presence of a single band around 70 kDa (Fig. 1, R3) in opposition to the multitude of high molecular weight proteins present in the crude extract (Fig. 1, R2) is a confirmation of the effectiveness of this purification procedure. The high hydrophobicity of S. lycocarpum PPO and the presence of protein subunits were confirmed by treatment of the partially purified enzyme with Triton X100 (Fig. 1, R4), that improved the proteins electrophoretic mobility, evidencing the presence of two bands around 47 kDa and 68 kDa. There are reports in the literature that PPO from vegetable sources present molecular weight varying from 39 kDa to 220 kDa [1,3]. This purification process was quite remarkable when compared to those described in the literature, especially considering its simplicity. The single step methodology of purification used in this

study was more effective than those presented by Marri et al. [2] for purification of PPO from potato tubers using a phenyl sepharose (fourfold); Pinto et al. [3] for purification of PPO from cowpea (3.3fold); and Guo et al. [1] for purification of PPO from beans using ammonium sulfate (3.5-fold).

3.3. Effect of pH and temperature on the PPO activity The enzymatic profile of the partially purified PPO from S. lycocarpum at different values of pH was shown in Fig. 2a. As can be seen, the partially purified PPO presented maximum activity in the range of pH 6.0–6.5. Although the activity decreases below and above 6.5, the enzyme was still active at pH 3.0 with a remaining activity of 31%. At pH 8.0, the remaining activity of enzyme was 26%. Several authors reported that the optimal pH for PPO activity varies from about 4.0 to 7.0 depending on the extraction methods, substrates and localization of the enzyme in the cell [30,31]. The temperature profile for the catechol oxidation by the partially purified PPO is presented in Fig. 2b. As observed, the optimum temperature of reaction was 28 ◦ C. The increase in the PPO activity as function of temperature could be due to the fact that the increase of temperature enhances the kinetic energy which leads to an acceleration of the reaction [32]. However, in the temperatures higher than 45 ◦ C was observed a reduction of 68% in the PPO activity, probably due to thermal denaturation of the enzyme.

Fig. 1. Electrophoresis (SDS-PAGE) of crude extracts and partially purified PPO from Solanum lycocarpum. R1: molecular markers; R2: crude extract of S. lycocarpum unripe fruits (40 ␮g protein); R3: partially purified PPO without Triton X100 (40 ␮g protein); R4: partially purified PPO with Triton X100 (20 ␮g protein).

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 211–217

215

Fig. 2. Effect of (a) pH and (b) temperature on the activity of Solanum lycocarpum PPO.

Table 3 Kinetic parameters for the thermal inactivation of Solanum lycocarpum PPO at different temperatures. Temperature (◦ C) 30 40 50 60 65

Kd (h−1 ) 0.13e 0.26d 0.33c 0.44b 0.89a

± ± ± ± ±

0.009 0.012 0.007 0.003 0.023

T1/2 (h) 2.71a 2.03b 1.82c 1.51d 0.80e

± ± ± ± ±

D (h) 0.07 0.05 0.02 0.01 0.03

17.31a 8.78b 7.08c 5.22d 2.57e

± ± ± ± ±

1.13 0.41 0.16 0.04 0.07

Results are presented as the mean ± standard deviation of three determinations. In the columns, the values followed by the same letter are not statistically different (p > 0.05).

3.4. Kinetic analysis of thermal denaturation A detailed kinetic study of isothermal inactivation of S. lycocarpum PPO was performed at different temperatures (30–65 ◦ C). The denaturation constants (kd ) were calculated and are given in Table 3. The rate constant increased with the heating temperature, indicating that S. lycocarpum PPO activity decreases at higher temperatures. The half-life (t1/2 ) and the decimal reduction time (Dvalue) are other important parameters commonly used in the

characterization of enzyme stability. Increasing the temperature from 30 to 65 ◦ C resulted in a decrease in t1/2 and D-values (Table 3). Comparing the D and t1/2 values from partially purified PPO with those from other sources, PPO from S. lycocarpum was more thermostable than polyphenol oxidase from mushroom [18,33], apple [34], and durum wheat [35]. Differences in kinetic of heat activation of PPO from differences sources may be a result of the differences in the plant variety as well as agronomic and climatic conditions under which they were grown [35,36]. A linear relationship was observed in the plot of log D versus temperature (r2 = 0.99). From this plot, the z-value was calculated as 49 ◦ C for S. lycocarpum PPO. In general low z-values are thought to indicate the sensitivity to heat. According to ´ Vámos-Vigyazó and Haard [37], z-values for thermolabile PPO vary from 8.5 to 10.1 ◦ C, while z-values above 30 ◦ C are commonly related to thermostable PPO. In this sense, considering the z-value, the S. lycocarpum PPO can be considered as a thermostable enzyme. The dependence of the inactivation constants with temperature was adequately fitted by Arrhenius equation (r2 = 0.92). This linearity is an indicative that the inactivation of the S. lycocarpum PPO occurs through a unique mechanism dependent on the temperature.

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Table 4 Thermodynamic parameters for the thermal inactivation of PPO from Solanum lycocarpum at different temperatures. Temperature (◦ C) 30 40 50 60 65

H (kJ mol−1 )

G (kJ mol−1 )

± ± ± ± ±

d

a

37.39 37.31a 37.22a 37.14a 37.10a

0.93 0.98 0.99 0.93 0.90

104.47 106.24c 109.14b 111.76a 111.49a

± ± ± ± ±

0.16 0.12 0.06 0.02 0.07

S (J mol−1 ) −221.40 −220.24a −222.66a −224.08a −220.10a a

± ± ± ± ±

2.87 3.16 3.17 2.94 2.93

Table 5 Substrate specificity and kinetic parameters of polyphenol oxidase from Solanum lycocarpum fruits. Substrate Catechol 4-Methylcatechol Pyrogallol Phenol

Specific activity (U mg−1 protein) 20.97b 67.18a 3.82c 1.78c

± ± ± ±

1.74 3.00 1.19 0.84

Vmax (U mL−1 )

Km (mM)

3.42 3.01 3.90 –

6.47 0.15 20.11 –

Results are presented as the mean ± standard deviation of three determinations. In the columns, the values followed by the same letter are not statistically different (p > 0.05).

Results are presented as the mean ± standard deviation of three determinations. Within column, values followed by the same letter had no significant difference (p > 0.05).

From 30 ◦ C to 65 ◦ C, the activation energy (Ea ) value for thermal inactivation of PPO from S. lycocarpum was calculated to be 39.9 kJ mol−1 . This Ea value was lower than those presented by Dincer et al. [38] for PPO from medlar fruits; Gouzi et al. [18] and Liu et al. [33] for mushroom PPO; Gnangui [39] for edible yam; and Waliszewski [40] for vanilla bean. High activation energy reflects a greater sensitivity of PPO to temperature change. On this basis, the PPO from S. lycocarpum can be classified as a heat resistant PPO in terms of inactivation kinetic.

similar (p = 0.001) in the studied temperatures. This result suggests a decrease in disorder or randomness of the enzyme/solvent system upon denaturation. The most common cause of the heat inactivation of enzymes is the loss of native conformation, a process identified as thermodenaturation, which takes place as a result of increased molecular mobility at elevated temperature. Therefore, considering the S values, the S. lycocarpum PPO can be considered a heat-resistant enzyme. In addition, as reported by Olusesan et al. [43], positive S values are found if the rate-limiting reaction is the protein unfolding, as result of moderately high values of H and low values of G. On the other hand, negative S values are result of moderately low values of H and high values of G. The negative S value observed for the inactivation of S. lycocarpum PPO indicates that the rate-limiting reaction probably involves the aggregation of partially unfolded enzyme molecules which predominate during the exposure of protein to high temperatures.

3.5. Thermodynamic analysis of thermal denaturation The determination of the thermodynamic parameters of inactivation provides information regarding to enzyme thermostability for each step of heat-induced denaturation process [18]. Moreover, these parameters may help in detecting the possible effects of secondary stabilization or destabilization of the tridimensional structure of an enzyme. The thermodynamic parameters G, H and S were calculated in the temperature range of 30–65 ◦ C and are depicted at Table 4. G is the Gibbs free energy change considered as the energy barrier for enzyme inactivation; H is the enthalpy change measuring of the number of bound broken during inactivation; and S is the entropy change that indicates the net enzyme and solvent disorder [18]. The multivariate analysis of enthalpy results showed that H values in the studied temperatures was not statistically different. This fact evidences that there is no change in enzyme heat capacity (Table 4). The H value for S. lycocarpum PPO was higher than those reported by Colak et al. [41] for mushroom PPO, and lower than those described for other PPO [18,33,38]. In general, H is seen as a measure of the number of noncovalent bounds broken in forming a transition state for enzyme inactivation. However, the use of isolated H values as indicators of enzyme stability is not suitable [42]. In fact, the stability of a protein is the result of a delicate balance between stabilizing and destabilizing forces, which are influenced by the number of disulfide and hydrogen bonds, the folding degree and hydrophobicity of the molecule, and the total of ionic and other interactions. Therefore, the protein stability is directly related to G values where the higher the G, the higher will be the enzyme stability. When the incubation temperature was elevated from 30 to 60 ◦ C, there was a significant increase of G values for the S. lycocarpum PPO, indicating that there was not destabilization of this protein with the increase in the heat temperature. This behavior is similar to those observed for PPO from other plants [38–41]. In addition, all G values were in agreement with the relative constant value of 100 kJ mol−1 characteristic of the protein denaturation reaction as already found to other PPO [18,33,39,40]. Since G increases with increasing temperature whereas H is overall constant, one could expect a significant contribution of entropy changes to the thermodynamics of the studied system. As can be seen in Table 4, all S values for thermal inactivation of the PPO from S. lycocarpum are negative and statistically

3.6. Substrate specificity and kinetic parameters A variety of phenolic compounds has been used as substrates of PPO in the literature [1,2,27,35,44]. In this study, the substrate specificity of PPO was examined using monophenols, diphenols, and triphenols substrates (Table 5). As can be observed, the PPO was most active with the o-diphenol 4-methylcatechol and catechol, which evidenced a catecholase activity. However, there was no significant activity toward pyrogallol and phenol, suggesting the absence of cresolase activity. The values of the kinetic parameters (Km and Vmax ) for catechol, 4-methylcatechol and pyrogallol are shown in Table 5. The partially purified PPO has a high affinity toward 4-methylcatechol, which was the best substrate of those tested (lowest Km value). The differences in the specificity of PPO against the various substrates can be explained by the tridimensional organization of the enzyme catalytic site. The chemical structures of catechol and 4methylcatechol are similar, composed by substituted benzene at the ortho and meta positions. However, the presence of a methyl group in the 4-methylcatechol structure replacing the hydroxyl presented in catechol, may be the most important factor for the different specificity observed. Regarding to phenol, the absence of the cresolase activity impede the further oxidation of this compound. Otherwise, the presence of an additional hydroxyl group in the structure of pyrogallol probably interferes in the interaction between enzyme-substrate. 3.7. Inhibition studies The effects of inhibitors on S. lycocarpum PPO activity were investigated in this study. The percentage, mode of inhibition and the values of inhibition constants (Ki ) are given in Table 6. Among the tested molecules, l-cysteine was found to be the inhibitor with the higher percentage of PPO inhibition (74.5%), followed by sodium metabisulfite (70.5%), ascorbic acid (70.3%), thiourea

K.A. Batista et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 211–217 Table 6 Effect of inhibitors on the PPO activity and type of inhibition.

References

Inhibitor

I (mM)

% Inhibition

Ki

Inhibition type

Ascorbic acid Citric acid Thiourea Sodium metabissulfite Cistein

30 10 15 10 10

70.3 24.3 46.0 70.5 74.5

3.98 1.24 1.18 1.62 3.25

Uncompetitive Uncompetitive Uncompetitive Uncompetitive Competitive

(46.0%) and citric acid (24.3%). The type of inhibition was competitive for l-cysteine and uncompetitive for all others. Among the uncompetitive inhibitors, the thiourea showed the lower Ki value (1.18 mM), followed by citric acid (1.24 mM), sodium metabisulfite (1.62 mM) and ascorbic acid (3.98 mM). The variation observed in the percentage of PPO inhibition and Ki values are related to differences in the mechanism of inhibition, depending on the compound used. Inhibition of PPO by l-cysteine has been attributed to the stable colorless products formed by reaction of this compound with o-quinones [35]. Similarly, the sodium metabisulfite can act as a reducing agent for o-benzoquinones, while thiourea can react with quinones, interfering with PPO activity. Ascorbic acid acts more as antioxidant than as enzyme inhibitor because it reduces de initial o-quinone formed by the enzyme to the original diphenol before it undergoes secondary reaction which leads to browning. Finally, the inhibitory effect of citric acid probably is due to its binding with active site copper, to form an inactive complex [45]. 4. Conclusions In this work, polyphenol oxidase from S. lycocarpum was extracted and partially purified by a single step freezing precipitation, resulting in a sixfold purification. Results evidenced that the unripe fruits presented PPO activity about fivefold higher than the ripe fruits. The partially purified PPO presented catecholase activity toward catechol and 4-methylcatechol, and the optimal activity conditions of the enzyme were determined as 28 ◦ C and pH 6.5. The enzyme appears to have biochemical characteristics similar to several plant PPO in terms of substrate specificity, pH and temperature of assay and kinetic parameters. Moreover, S. lycocarpum PPO was sensitive to some general PPO inhibitors, especially to l-cysteine, sodium metabisulfite and ascorbic acid. Results from thermal inactivation of S. lycocarpum PPO at 30–65 ◦ C evidenced half-life (t1/2 ) varying from 2.71 to 0.8 h and Dvalues between 17.31 and 2.57 h. The thermodynamic parameters suggested that the partially purified PPO is a temperature-resistant biocatalyst, whose inactivation process is related to aggregation of partially unfolded enzyme molecules. Finally, based on the results obtained in this work, and considering the high productivity of S. lycocarpum, it is possible to indicate S. lycocarpum fruits as a promising source of polyphenol oxidase for biotechnological applications that demands a thermostable catecholase activity. Acknowledgement K.A. Batista thanks CAPES for fellowship support.

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