properties of polyphenoloxidase and peroxidase from granada · of food technology 233 autores...

OF FOOD TECHNOLOGY

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AUTORESAUTHORS

RESUMO

PALAVRAS-CHAVEKEY WORDS

SUMMARY

Properties of Polyphenoloxidase and Peroxidase from Granada Clingstone Peaches

1 Departamento de Ciência e Tecnologia de Alimentos,

Universidade Federal de Pelotas,

CP 354, CEP 96010-900, Pelotas, RS, Brazil,

e-mail: [email protected]*EMBRAPA Clima Temperado,

KM 78, BR 392, CP 403;

CEP 960001-970, Pelotas, RS, Brazil;

e-mail: [email protected] de Biotecnologia,

Universidade Federal de Pelotas, Pelotas, RS

e-mail: [email protected] Bioquímica,

Universidade Federal de Pelotas, Pelotas, RS5Departamento de Ciência e Tecnologia de Alimentos,

Universidade Federal de Pelotas, Pelotas, RS

1 Ricardo Peraça TORALLES2*João Luiz VENDRUSCOLO3Claire Tondo VENDRUSCOLO4Francisco Augusto Burkert DEL PINO5Pedro Luiz ANTUNES

Estudou-se o escurecimento enzimático objetivando definir a enzima mais termoestável responsável pelo escurecimento na cv. Granada, que é amplamente cultivada no Sul do Rio Grande do Sul, Brasil. A polifenoloxidase (PPO) e peroxidase (POD) foram extraídas da cultivar de pêssego Granada usando o método do pó-de-acetona, purificação parcial pela precipitação com (NH ) SO seguido de diálise. As ótimas condições de reação foram 4 2 4

estudadas usando catecol como substrato para PPO e guaiacol: H O para POD. O pH ótimo foi 2 2

6,2 em catecol e 5,0 guaiacol:H O respectivamente. A temperatura ótima para máxima 2 20atividade foi 30 C e ambas enzimas foram afetadas pela desnaturação térmica, sendo a PPO

mais termoestável. A PPO e POD de pêssego Granada mostraram atividade com todos substratos o-difenólicos: catecol, pirogalol, 4-metil-catecol e L-dopa. Além disso, POD oxidou o guaiacol na presença de peróxido de hidrogênio, mas a PPO não foi capaz disso. O K foi 9,7 M

mM de catecol para PPO e 5,2 mM de guaiacol para POD. Na eletroforese, duas isoenzimas foram detectadas com os substratos catecol e guaiacol:H O .2 2

The enzymatic browning was studied aiming at defining mainly the most thermal- stable browning enzyme in the cv. Granada that is widely cultivated in the South of Rio Grande do Sul, Brazil. The polyphenoloxidase (PPO) and peroxidase (POD) enzymes were extracted from cv. Granada clingstone peaches using acetone powder method, partially purified by (NH ) SO 4 2 4

precipitation and dialysis. The optima conditions for reaction were studied using catechol as substrate for PPO and guaiacol: H O for POD. Optimum pH were 6.2 and 5.0 on substrates 2 2

catechol and guaiacol: H O respectively. The optimum temperature for maximum activity was 2 2030 C and both enzymes were affected by the heat denaturation, being PPO more thermostable.

The Granada peach PPO and POD showed activity with all the o-diphenolic substrates: catechol, pirogallol, 4-methyl-catechol and L-dopa. In addition, POD can also catalyze the oxidation of guaiacol in the presence of hydrogen peroxide, whereas PPO was unable to do so. The K was 9.7 mM catechol for PPO and 5.2 mM guaiacol for POD. In electrophoretic M

separation, two isoenzymes were detected with catechol and guaiacol:H O substrates.2 2

Pêssego Granada, escurecimento enzimático,

parâmetros de Arrhenius e isoenzimas.

Granada peach, enzymatic browning,

Arrhenius parameters and isoenzymes.

Braz. J. Food Technol., v.8, n.3, p. 233-242, jul./set. 2005

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1. INTRODUCTION

2. MATERIALS AND METHODS

About 50,000 tons of clingstone peach is canned every year in Southern Brazil. Now, the processors are looking for high aggregated value and innovative products for the Brazilian market. High quality peach purée is a commodity requested by manufactures of drinks, jellies, ice cream and others. Granada clingstone peach is an early cultivar, double purpose type and represents about 25% of the total cultivated area.

Maintenance of natural color and flavor are a challenge in the processing of peach in syrup and mainly peach purée, pulp and juices (LUH, 1980; ASHURTS, 1995). Enzymatic browning reactions are a widespread phenomena that induce severe color changes, undesirable flavors and nutritional losses (CHUNG and LUH, 1972; VÁMOS-VIGYÁZÓ, 1981; GIRNER et al., 2002). The polyphenoloxidase (1,2-benzenediol:O oxidoreductase; EC 1.10.3.1; PPO) and 2

peroxidases (donor:hydrogen-peroxide oxidoreductase; E.C. 1.11.1.7; POD) constitute an important group of enzymes associated with the enzymatic browning of phenolic compounds, which are found in pulps of fruits and are widely distributed in plant tissues (FLURKEY and JEN, 1978; DUPUY, 1982). PPO enzymes catalyze the oxidation of phenolic substrates using oxygen as hydrogen acceptor in two very different types of reactions. These reactions involve hydroxylation of monophenols to give o-diphenols and the removal of hydrogens from o-diphenols to give an o-quinone (WHITAKER, 1994). In the clingstone peach has been reported activity on o-diphenols exclusively and no ability to hydroxylate monophenols ( LUH and PHITHAKPOL, 1972).

Peroxidase catalysis is associated with four types of activity: peroxidatic, oxidatic, catalatic, and hydroxilation. For phenolic substrate, only the peroxidatic reaction is of importance. The action of the enzyme is primarily aimed at controlling the level of peroxides, which are generated in almost every reaction in living cells and in the absence of a hydrogen donor. Peroxidase converts hydrogen peroxide to H O and O in a catalatic reaction. This reaction is at least 1000 2 2

times slower than the peroxidatic ( RANIERI et al., 2001; WHITAKER, 1994). Recently, POD has gained much attention due to its capability to generate phenolic crosslinks connecting neighbouring biopolymer chains (BARCELÓ, 1998; TIJSKENS et al., 1997) and that cell-wall polysaccharides can be depolymerized by POD acting in the OH-generating (SCHWEIKERT, LISZKAY and SCHOPFER, 2000; SCHOPFER, 2001).

These enzymes are known since long date in peaches and continue being studied (REYS and LUH, 1960; FLURKEY and JEN, 1978; ALBA et al., 1996; GIRNER et al., 2002) and others plant tissues, including nectarine (CHENG and CRISOTO, 1995), apples (WALKER and WILSON, 1975; GOUPY et al., 1995); custard apples (LIMA et al., 2001), pears (LUH et al., 1963) and broccoli (FUNAMOTO et al., 2003). However there are few studies published on heat inactivation of POD from peach (TIJSKENS et al., 1997; NEVES, 2002) and the inactivation parameters for peach PPO were not found.

Little has been reported about the enzymatic browning in Brazilian clingstone peaches. In Granada clingstone peach a low value V /K -PPO, called the specificity max m

coefficient for PPO, was observed among three other Brazilian clingstone peach cultivars (TORALLES et al., 2004). This coefficient is a good indicator of enzymatic browning in vitro. The knowledge of the kinetic resolution will allow knowing the potential of these cultivars for the processing of products such as juices, pulps and preserves. This fact besides its technological importance, also presents significant economical dimensions, because it affects the quality and competitiveness of the products.

In the present work, the parameters related to the enzymatic browning in Granada clingstone peaches and its thermal control were investigated. With this purpose, characteristics and behavior of PPO and POD at different pH, temperature, phenolic substrates and substrate concentration were studied. Three parameters, the inactivation rate constant (k) at a reference temperature, the activation energy for inactivation (Ea) and half-lives (t ) were determined for each 1/2

enzyme to define the most thermal-stable browning enzyme in the cv Granada.

2.1 Raw fruit

Mature and sound appearance peaches clingstone cv. Granada were picked from a commercial orchard in

0Pelotas/RS and stored in cold room between 1and 3 C for a maximum of 2 days during the assays.

2.2 Physical and chemical Properties

Fifteen peaches were divided into three replications of five fruit each. The pit was removed and the slices were ground to a juice in a centrifugal grinder. Duplicate analyses were carried out. Soluble solids (SS), pH, titratable acidity (TA), reducing sugars, and total sugars were determined in the fresh juice using AOAC (2000) methodology. Total phenolics were determined by the method SINGLETON and ROSSI (1965). The external color of the fruit was measured with a Minolta colorimeter model CR 300 (Japan), calibrated with plate (L= 96.98, a=+0.26, b=+1.78). The color data were converted to

-1hue angle = tan (b/a) (MEREDITH, ROBERTSON and HOVART, 1989). Firmness was measured on two sides of the 15 peaches using a pressure penetrometer with an 8-mm plunger.

2.3 Preparation of acetone powder

The first step in purification is the cells and membranes breakage and extraction of enzyme from the plant tissues. The preparation of acetone powders is the usual (WHITAKER, 1994). Fresh unpeeled peaches were pitted, quartered, and quickly frozen. The frozen pieces were milled

0with acetone at -20 C in an electric mill (Wallita,Brazil) for 5 minutes. A ratio of 2 liter of cold acetone (Synth, Diadema/SP, Brazil) per kilogram of milled frozen peach was used. The resulting mixture was suction filtered through paper Whatman No. 1. The obtained powder was dried overnight at refrigerator temperature to remove residual acetone and it was called


TORALLES, R. P.; VENDRUSCOLO, J. L.; VENDRUSCOLO, C. T.; DEL PINO, F. A. B.

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1.

kt- )A

Aln( or (k/2.303)t)

A

Alog(

o

t

o

t =-=1.

kt- )A

Aln( or (k/2.303)t)

A

Alog(

o

t

o

t =-=

lnAEa/RTln(k) +-=2.

Granada-peach-acetone-powder (GPAP). This was filled into plastic flasks, stamped in inert atmosphere of N and stored at -2

020 C.

2.4 PPO and POD extract preparation

0All stages were carried out below 5 C. The GPAP (3.3 g) was homogenized with 100 mL of chilled 100mM citrate-200mM phosphate buffer (citric acid and K HPO 2 4

purchased from Merck, Darmstadt, Germany) in pH=6.2, which contained 3.3g of PVP (Sigma-Aldrich, Steinhelm, Germany) and 1M of KCl (Synth, Diadema/SP, Brazil); all was stirred together in a glass vessel for 20 minutes. The homogenate was centrifuged at a relative centrifugal force of 20,000xg for 20 minutes with Avanti j-25 centrifuge (Beckman Instrument Inc., Fullerton, Calif., U.S.A). The supernatant was filtered through Whatman No.1 paper and brought to 80% (NH ) SO saturation. The precipitated part was separated by 4 2 4

centrifugation at 20,000xg for 20 minutes. It was also odissolved in homogenization buffer and dialyzed at 4 C

overnight. These extracts were distributed in tubes of 10 mL, 0sealed and frozen at -20 C. Protein determination was made

by method of LOWRY et al. (1951) and using BSA as standard (Acros, New Jersey, USA). The dialyzed sample was used as the enzyme source in the following experiments.

2.5 Assay of polyphenoloxidase and peroxidase activity

PPO activity was determined by measuring the increase in absorbance at 420 nm using a spectrophotometer Genesys 10 UV/VIS. PPO reacting mixture contained 9.0 mL of catechol substrate in the homogenization buffer (100mM citrate-200mM phosphate buffer) and 1mL of enzymatic extract. In the final reaction mixture, the concentration was 27.3 mM of catechol. The reaction was conducted at pH 6.2

oand 30 C. Catechol was used as a substrate except for the studies on phenolic substrates.

The spectrophotometric method was also used for measuring POD activity at 470 nm. Guaiacol, in the presence of hydrogen peroxide, was used as the substrate instead of catechol. POD reacting mixture contained 9.0 mL of guaiacol:H O in the homogenization buffer and 1.0 mL of the 2 2

enzyme. In the final reaction mixture, the concentration was 25.8 mM of guaiacol:H O . The reaction was conducted at pH 2 2

o5.0 and 30 C. Guaiacol was used as a substrate except for the studies on phenolic substrates. For blank, 1mL of homogenization buffer was used for both enzymes. Enzyme activity was calculated from the linear portion of the curve. One unit of enzymatic activity was defined as an increase of 0.01 unit of absorbance per minute and per milliliter of enzyme extract. The protein content of the enzymatic extract was 250 g per mL.

2.6 pH and temperature optima

The effect of pH on PPO and POD activity was oexamined at 30 C in the pH range 3 to 9, using appropriate

buffers (100 mM citrate-200 mM phosphate and 100 mM Tris-HCl). The determined optimum pH was used in all others

experiments. The temperature dependence of the PPO and 0POD activity was measured at temperature range 5-70 C using

circulator bath (Marconi, Piracicaba, Brazil). The reacting mixture and enzyme were heated up to the tested temperature and the enzyme was added.

2.7 Effect of substrate concentration and kinetic parameters

Catechol and guaiacol at various concentrations were used as substrate for peach PPO and POD respectively. In the final reacting mixture, the concentration ranged from (050mM) for both. The Michaelis constant (K ) and maximum M

velocity (V ) were obtained by non linear regression, and its max

points were confirmed by the linearity of the LINEWEAVER and BURK (1934) plot.

2.8 Phenolic substrates

The effect of phenolic substrate on PPO and POD activity was also studied. The phenolic compounds compared were catechol, pirogallol, 4-methyl-catechol, L-dopa and guaiacol. The activity for both enzymes was measured using the procedure described previously (item 2.5) with the following modifications: the final concentration of each phenolic substrate in the reacting mixture was 10 mM.

2.9 Heat stability, Arrhenius parameters and half-lives

Aliquots (500 L) of a solution of the enzyme (500 g/mL) in the homogenization buffer were heated in sealed glass tubes (i.d 9mm, wall 1mm) at different temperatures (40 to

o75 C) for different periods of time. After heating, samples were cooled on ice water bath and the remaining activity was measured using the procedure described previously with the following modifications: aliquots of 500 L of enzymatic extract were added in reacting mixture contained 4500 L of substrate. In the final reaction mixture, the concentrations were 27.3 mM for catechol and 25.8 mM guaiacol:H O . In this manner, 2 2

thermal denaturation of PPO and POD were monitored as related to heating time. The rate constants k for fist-order denaturation were determined from the slopes of the denaturation time courses according to Equation 1

where A is the initial enzyme activity and A is the activity after o t

heating for the specified time. The slopes of these lines were determined by linear regression, and the calculated rate constants were replotted in Arrhenius plots. Activation energies (Ea) were calculated from the slopes of the Arrhenius plots of ln(k) versus 1/T according to Equation 2



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3.

kt 2ln21 =

3. RESULTS AND DISCUSSION

Parameter

Harvest date

Avg. Wt/fruit (g)

pH

Titratable acidity (TA), % citric acid

0Soluble solids (SS), % at 25 C

Ratio (SS/TA)

Sucrose (%)

Total phenolics (g/g)

Color (Hue angle)

Firmness (N)

aValue

11-05/12-06

115.93 +_ 10.53

3.69 +_ 0.04

0.74 +_ 0.04

10.37 +_ 0.83

14.05 +_ 1.12

7.46 +_ 0.21

191.01 +_ 44.52

78.29 +_ 0.10

27.27 +_ 0.12

TABLE 1 Physical and chemical characteristics of Granada peaches.

amean +_ standard desviation

-1 -1where R is the gas constant (8.314 J.mol .K ), T is the temperature in K and A is called the pre-exponential factor. Slopes were calculated by linear regression.

A useful indication of the rate of a first-order chemical reaction is the half-life t , of a substance, the time it 1/2

takes for its concentration to fall to half the initial value. The time for [A] to decrease from [A] to ½[A] in a first-order o o

reaction is given by Equation 1 as

Hence

The main point to observe about this result is that, for a first-order reaction, the half-life of a reactant is independent on its initial concentration.

2.10 Electrophoresis

PPO and POD enzyme system were subjected to horizontal polyacrylamide gel electrophoresis. Polyacrylamide gel (5%) and electrode buffer (pH=8.3) were prepared according to the method of SCANDALIOS(1969) to separate the isoenzymes of Granada peach. The enzyme sample (1000 g/mL) was loaded (40L) into each space. A voltage of 90-110V was applied in the strip of the separation gel for 4 hours. The

oelectrophoresis was carried out at 4 C. After running, the gel was cut into two symmetrical parts. One part was revealed PPO by immersion in the homogenization buffer in pH=6.2 containing 10 mM of catechol, and other part immersed in the homogenization buffer in pH=5.0 containing 10 mM guaiacol:H O . The isoenzyme bands were developed for 1 2 2

hour and each gel was washed with solution of ascorbic acid 1mM for 5 minutes. Afterwards, the gels were photographed.

2.11 Statistical analysis

The coefficient of each parameter in the linear model fits was determined by least square method using the MATHCAD software (Mathsoft,1995) statistical functions for linear regression of X-Y data. The method of Quasi-Newton was used for non-linear regression using the STATISTICA software (Statsoft, 1998). The two-dimensional plots, showing effects of each reaction conditions, were generated by MATHCAD software (Mathsoft, 1995) graphics plot. Where applicable, results were analyzed by the ANOVA.

ln2ln(1/2)21

lnk. t2

1 =

÷÷÷÷

ø

ö

çççç

è

æ

-=o

o

A

A-=

3.1. Physical and chemical characteristics of Granada peaches

The characteristics of the Granada cling peaches used in this investigation are shown in Table 1. The color and firmness are characteristic of ripe fruit (ROBERTSON and MEREDITH, 1988). The relatively low value for the ratio (SS/TA) and total phenols can be attributed to early season cultivar type. Its has been suggested that the ratio SS/TA indicates ripeness of fresh fruit and height-quality fruit, SS/TA 15.1(DESHPANDE and SALUNKHE, 1964). Mature Redhaven peaches (midseason cultivar) had minimum SS/TA value of 13 and is used for peach juice (ROBERTSON et al., 1990; VERSARI et al., 2002). Mature Cresthaven peaches (late-season) had SS/TA value of 23.1 and are known by high-quality fruit (ROBERTSON et al., 1990). Low total phenolics concentration is recommendable to attenuate the oxidative darkening phenolic substrates and is a good indicative of the potential of Granada peach to be processed.

3.2 pH and temperature optima

Figure 1 shows the e f fect o f pH on polyphenoloxidase and peroxidase activity. The pH optimum of PPO for the cathecol oxidation was at pH 6.0-6.5. Maximum POD activity for the guaiacol:H O oxidation was at pH 4.8-5.5. 2 2

At pH 3.8, characteristic value for Granada peach, peroxidase activity predominated over polyphenoloxidase activity. POD retained 43% of its maximum activity, whereas PPO retained only 8%. As Granada peach PPO and POD activity were pH dependent, this property is very important from the point of view of controlling enzymatic browning of peaches. The result presented here agrees with the observation of REYS and LUH (1960) to pH-dependence.



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0The optimum temperature was 30 C for both enzymes (Figure 2). The stability of the peroxidase was smaller

0than the one of the polyphenoloxidase starting from T = 40 C. This property is very important in the browning control of peaches.

3.3 Kinetic parameters

The kinetic parameters of catechol and guaiacol:H O oxireduction reaction were determined for PPO 2 2

and POD respectively. For PPO, the plot of the rate of oxidation reaction of catechol versus substrate concentration was hiperbolic in the first part of the curve, suggesting Michaelis-Menten kinetics (Figure 3). This point was confirmed by the linearity of the Lineweaver-Burk plot (Figure 3, insert). In second part, activity decreased for a catechol concentration higher than 38 mM. This inactivation may result from classical substrate inhibition.

When guaicol:H O was used as substrate, a large 2 2

plateau was observed for the POD, suggesting a better adaptation to the Michaelis-Menten kinetics (Figure 4). The plot of the rate of peroxidatic reaction versus substrate concentration was confirmed by the linearity of the Lineweaver-Burk plot (Figure 4, insert).

Kinetic parameters were estimated by non-linear regression (Table 2). Therefore, apparent Michaelis constants should be used. This precaution is necessary due to the partial purification of the enzymatic extract. The K value for PPO in M

Granada peaches was 9.7 mM catechol at pH=6.2. LUH and PHITHAKPOL (1972), working with canning-ripe Halford clingstone peaches, found that the Michaelis constant was 15 mM. Granada peach PPO shows a stronger affinity toward cathecol than Halford. The value V /K , called the specificity max m

-1 -1coefficients, was 228 for PPO and 1,400 U.mL .M for POD indicating that the peroxidase showed more significant activity



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Enzyme

PPO

POD

2R

0.99

0.99

aKm, app

(mM)

9.68 +_ 3.15

5.15 +_ 2.21

aVmax

-1(U.mL )

2.21 +_ 0.24

7.21 +_ 0.81

-1 -1(U.mL .M )

228

1,400

m

max

KV

TABLE 2 Michaelis constant (K ) and maximum velocity m, app

(V ) of PPO and POD of Granada clingstone peach.max

avalues +_ confidence interval at p=0.05

than polyphenoloxidase of Granada peach. According with TORALLES et al. (2004), the PPO-Granada showed a higher K m

and consequently a lower enzymatic browning capability among three other Brazilian clingstone peach cultivars. Also the value of larger K -PPO than of total phenolics (1.7 mM) of m

Granada peach is a good indicative of the low tendency to the darkening of the Granada peach that can be found in practice.

3.4 Phenolic substrates

The effect of phenolic substrates on PPO and POD oactivity at 30 C are shown in Table 3. The catechol was oxidized

by both enzymes, but with different rates. PPO activity was 1.1 -1U.mL , equivalent to 100% of relative activity (RA), and POD



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Substrate

(10mM at pH 6.2)

Catechol

4-methyl catechol

Pyrogallol

L-dopa

Guaiacol

PPO

(Relative activity %)

a100

46

60

41

0

Substrate

(10mM at pH 5.0)

Catechol:H O2 2

4-methyl catechol: H O2 2

L-dopa: H O2 2

Pyrogallol: H O2 2

Guaiacol: H O2 2

POD

(Relative activity %)

35

41

23

15

b100

TABLE 3 Effect of phenolic substrates on PPO and POD of Granada clingstone peach.

a o -1 b o -1 activity at 30 C = 1.1 U.mL / activity at 30 C= 4.8 U.mL

activity was 35% of RA. Among the substrates tested, peach PPO was the most active toward catechol, followed by pyrogallol, 4-methyl catechol and L-dopa. PPO from apple (OKTAY et al., 1995) has also been reported to catalyze the oxidation of these o-diphenolic substrates. Granada peach POD oxidized guaiacol in the presence of hydrogen peroxide,

-whereas PPO was unable to do so. POD activity was 4.8 U.mL1, equivalent to 100% of RA, followed by 4-methyl catechol, catechol, L-dopa and pyrogallol. In general it appears that the chemical configuration of o-diphenols is appropriate for peach PPO and POD substrates. In addition, POD can also catalyze the oxidation of phenols methyl-substituted, as found in guaiacol. These results show that activity parasite doesn't exist using the catechol as PPO substrate and the guaiacol:H O as POD 2 2

substrate.

3.5 Arrhenius parameters and half-lives

Thermal inactivation of POD in the guaiacol substrate showed apparent first-order kinetics (Figure 5), and its residual activity (Log %) was typically linear between 40-

o50 C. At the longest heating times, there is an apparent deviation from linearity. This deviation could indicate the presence of a second isoenzyme with greater thermal resistance. Tomato juice pectin methylesterase showed similar behavior (ANTHON et al., 2002).

Thermal inactivation of PPO in the catechol substrate also showed apparent first-order kinetics (Figure 6),

0 oand its activity decrease rapidly above 55 C. At 60 C, 80% of the activity was lost after 540 seconds and similar loss was

oobserved after incubation at 75 C for about 90 seconds, suggesting that PPO catalytical structure is changed significantly on increasing temperature. This probably happens because of the importance of noncovalent bond in maintaining



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3. CONCLUSIONS

the structure of enzymes. These bonds involve van der Waals forces, eletrostatic interactions, hydrogen bonds and hydrophobic interactions (WHITAKER, 1994). When high temperatures disrupt these nonconvalent interactions, proteins unfold (VIEILLE, ZEIKUS, 2001). Similar behavior was observed

0for POD, but its activity decreases rapidly above 45 C (Figure 5), indicating smaller POD thermostability than PPO.

The magnitudes of the Arrhenius parameters in Equation 2 determined by linear regression analysis are in Table

24. The coefficients of determination (R ) was of 0.993 for PPO and 0.994 for POD. Activation energy (E , i.e., the energy a

barrier to unfolding) and inactivation rate (k ) of POD were ref

higher than the PPO. In general, a higher activation energy implies that a smaller temperature change is needed to inactive an enzyme.

The results of TIJSKENS et al. (1997), working with thermal inactivation of peach POD (cv. Andross), are in good agreement with ours. They published a rate constant for

o -3 -1inactivation at 60 C of 8.44 x 10 s , and an activation energy of -1approximately 150 kJ.mol . According to NEVES (2002), an

-1activation energy of approximately 171 kJ.mol was found for bound POD from cv. Rei of Conserva peach.

In contrast with activation energy of the peach POD, the early results of ANTHON et al. (2002), working thermal inactivation of tomato enzymes, are larger. They report

0 -3 -1a rate constant for inactivation at 70 C of 32.4 x 10 s , and an -1 energy activation of 557 kJ.mol to POD in tomato juice from

the BOS 3155 cultivar. The energy activation and inactivation rate of POD were higher than pectin methylesterase and polygalacturonase.

The Arrhenius parameters values are, generally, not published for peach PPO, therefore comparisons are not possible. However, the energies of activation for heat-inactivation of PPO from different apple cultivars were studied by YEMENICIOGLU et al. (1997). They revealed that PPO in

-1Amasya (E = 255.6 kJ.mol ) was the least heat-stable and a-1Starking Delicious (Ea=240.6 kJ.mol ) was the most heat-

stable.

E n z y m e t h e r m o s t a b i l i t y e n c o m p a s s e s thermodynamic and kinetic stabilities. An enzyme's kinetic stability is often expressed as its half-life (t ) at defines 1/2

temperature (VIEILLE and ZEIKUS, 2001). Through the half-life it is possible to prove that PPO is kinetically more stable than

oPOD, because PPO half-life is three times POD half-life at 60 C (Table 4) and this behavior was observed in other temperatures (Figure 7). Peroxidase is generally one of the most thermally stable enzymes found in fruits and vegetables, it is a commonly used indicator for the inactivation. Thus, POD would not be a good indicator for enzyme inactivation in peach. Hereafter, the question now is about the nature of the structural changes between active and inactive forms of the PPO and POD, and how these might be determined.

3.6 Electrophoresis

Two isoenzymes for PPO and two for POD were separated by polyacrylamide gel electrophoresis and detected using catechol and guaiacol:H O respectively as substrates 2 2

(Figure 8). For PPO, a similar activity was observed between two bands (color intensity). For the POD one band with 0.4 of relative mobility was detected and this band is composed of two parts. Previous studies indicated from two to three POD isoenzymes that were found in different peach cultivars (AGARWAL et al., 2001). Comparative future studies can relate those isoenzymes with enzymatic browning.

PPO and POD of Granada clingstone peaches oshowed optimum activity at 30 C with different pH optima.

Heat treatments significantly reduced peach PPO and POD activity. The effectiveness of heat denaturation was quite high

oabove 70 C. The thermal stability of PPO was higher than that of POD and thus would be a good indicator for browning enzyme inactivation in peaches, but Granada peach POD presented higher activity than PPO. In both cases, heat denaturation was described using an exponential decay (first order reaction) and temperature dependence of all reaction was described by Arrhenius' law. Total phenolics smaller than K M

can be the explanation for the low browning observed in Granada peach. Two isoenzymes for PPO and POD were detected. Based on those physical-chemical characteristics and



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4. REFERENCES

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The authors thanks to State Department of Development and Foreign Affairs (SEDAI) for its financial support and CAPES for doctorate scholarship.

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