Effect of temperature on guaiacol Peroxidase of Pyrus communis

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase, POD) is one of the key enzymes controlling plant growth, differentiation and development. The enzyme participates in construction, rigidification and eventual lignification of cell walls, biosynthesis of ethylene from 1-aminocyclopropane-1-carboxylic acid and H 2 O 2 , regulation of auxin level through auxin catabolism, protection of tissue from damage and infection by pathogenic microorganisms, the oxidation of indoleacetic acid. For peroxidase activity in wild pears extract one pH optimum was observed at 6.5 that probably belong to atleast one isoenzyme. Activity of peroxidase in presence of guaiacol and H 2 O 2 was optimum after incubation at 40 °C. Maximum activity of peroxidase is 300 % .Activity increased to 240 %, 300 %, 70 % and 10 % after 60 minute incubation at 30, 40, 45 and 60 °C for peroxidase. Incubation at high temperature (70 °C) was accompanied with decrease of activity to 10 % peroxidase activity.


INTRODUCTION
Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase, POD) is one of the key enzymes controlling plant growth, differentiation and development. The enzyme participates in construction, rigidification and eventual lignification of cell walls, biosynthesis of ethylene from 1-aminocyclopropane-1-carboxylic acid and H 2 O 2 , regulation of auxin level through auxin catabolism, protection of tissue from damage and infection by pathogenic microorganisms, the oxidation of indoleacetic acid (Dunford 1991; Wakamatsu and Takahama, 1993). In vitro, this enzyme is widely employed in microanalysis (Krell, 1991). More than 80 % of immunoenzymatic kits contain peroxidase as labeling enzyme. Recently, peroxidases have been used for biotransformation of organic molecules (Adam et al., 1999). It has been well established that peroxidase as one of the most stable enzymes can contribute to deteriorative changes in quality of the processed products (Stanley et al., 1995). Enzymatic browning is one of the most important colour reactions that affect fruits and vegetables.
It is catalyzed by polyphenoloxidases and peroxidases (Jiang et al., 2004). It was estimated that over 50 percent losses in fruits and vegetables occur as result of enzymatic browning (Whitaker and Lee, 1995). Marshall et al. (2000) reported that the rate of enzymatic browning in fruits and vegetables is governed by the active polyphenoloxidase and peroxidase content of the tissues, the phenolic content of the tissues, pH, temperature and oxygen availability with the tissue. According to Khan and Robinson (1993) the peroxidase is directly responsible for enzymatic browning in mangoes. The objective of this work was to study the effect of pH and heat on peroxidase activity and to extend the knowledge of vegetables peroxidase in particular.

MATERIALS AND METHODS
For extract preparation, wild pears were used throughout these studies. Extracts were prepared from pears weighting each between 50 and 80 g by homogenization in phosphate buffer 0.1 M, pH 7 in presence of PMSF 2 %. After centrifugation at 3,000 g for 10 min, then at 35,000 g for 30 min, a clear, transparent supernatant termed "crude extract" was obtained and used for our studies. Protein concentration was determined by the Lowry method (Lowry et al.,1951).

1. Effect of pH
pH profile of peroxidase activities was determined spectrophotometrically at 25 °C by measuring the appearance of reaction products in the medium. The activity of the enzyme in wild pears were determined in the pH range 3-10 by using a citrate-phosphate-borate buffer 0.1 M. The optimum pH for PPO activity of extracts was obtained in presence of guaiacol (20 mM) and H 2 O 2 (10 mM) as substrates.
Enzymatic activity was determined by measuring the increase in absorbance at 420 nm with a spectrophotometer (6305 JENWAY). Assays were carried out by addition of 200 µl of extracts to the sample cuvette, and changes in absorbance were recorded. The reference cuvette contained just 3 ml of substrate solution. Enzyme activity was calculated from the linear portion of the curve. One unit of PPO activity was defined as the amount of enzyme that produces 1 micromole of product per minute. Assays were carried out at room temperature and results are the averages of at least three assays and the mean and standard deviations were plotted.

Effect of Temperature on peroxidase activity in wild pear
pH profile of Pyrus communis Peroxidase led to a peak at 6.5 that probably belong to atleast one isoenzyme. For determining the optimum temperature values of the enzyme, peroxidase activity was measured, at constant temperatures (27, 30, 40, 45, 50, 60 and 70 °C separately) using guaiacol (200 mM) and H 2 O 2 (10 mM). The effect of temperature on the activity of peroxidase was tested by heating the crude extract to the appropriate temperatures in different times.
The desired temperatures were provided by using a Memmert model ST/70 temperature controller attached. After different times (0-60 minute) at a same temperature, enzyme cooled in ice and was added and the reaction was followed spectrophotometrically at given time intervals as described above.

3. Heat Inactivation of peroxidase
The thermal denaturation of the peroxidase was studied at different temperature solution in a test tube was incubated at the required temperature for fixed time intervals. At the end of the required time interval, the test tube was cooled in an ice bath. The activity of the peroxidase was then determined.

1. pH profile of peroxidase activity in Pyrus communis
pH is a determining factor in the expression of enzymatic activity; it alters the ionization states of amino acid side chains or substrate. For peroxidase activity, one pH optimum were observed at 6.5. No activity was detectable at pH 3 and 9, regardless of the condition. Figure 1 shows the pH activity profile obtained for peroxidase.

2. Effect of temperature on peroxidase activity
Time courses at 30 ºC showed an increase of peroxidase activity so, after 60 minute incubation, activity reached to 240 % (Fig. 2). Time course at 40 ºC has the similar condition, so increase in time of incubation accompanied with more increase in activation. After 60 minute incubation of peroxidase in 40 ºC, activity reached to 300 %. Time course at 45 ºC has the different results, so increase in time of incubation accompanied with more increase in activation up to near 20 minute. Increase in time of incubation more than 20 minute up to 60 minute accompanied with decrease in activation, so after 60 minute incubation of peroxidase in 45 ºC, activity decreased to 70 %. Time course at 50 and 60 ºC showed different behavior for peroxidase, so increase in time of incubation accompanied with more decrease in  We can conclude that increase in temperature from 30 to 40 ºC change probably structure of peroxidase to ordered shape, so that activity of enzyme in 30 and 40 ºC increased and in more temperature (60 and 70 ºC) enzyme slowly denature and activity decreased. Activity of peroxidase after 60 minute incubation at 40 ºC is 1.5, 5 and 33 times more than 30, 45 and 60 ºC.

Optimum Temperature and Thermal Inactivation
PPO in presence of guaiacol and H 2 O 2 showed fluctuations in activity with increasing temperature even as high as 70 °C. The plot for guaiacol demonstrated that the enzyme was very thermostable between 25 and 45 °C. Our results showed optimum temperature is 40 °C for both peroxidase activity. Like most chemical reactions, with increase of temperature from 27 ºC, gradually, activity of peroxidase increased so; we reached to maximum of activity at 40 ºC (300 %) (Fig. 3). With more increase in temperature from 40 ºC to 70 ºC, activity of peroxidase decreased gradually and reached to 10 % at 70 ºC. The drop in percentage residual activity at high temperatures can actually be due to the unfolding of the tertiary structure of the enzyme to form the secondary structure. Activity of peroxidase even in 70 ºC was observed and is 10 % related to control (25 ºC). These results showed that, peroxidase show activity at high temperatures. A ten degree Centigrade rise from 40 to 50 ºC in temperature after 5 minute incubation will increase the activity of peroxidase from 170 to 70 %. Variations in reaction temperature as small as 10 degrees from 30 to 40 ºC introduce increase of 50 % in the activity and from 60 to 70 ºC introduce decrease of 50 % in the activity.

CONCLUSION
For peroxidase activity, this is complicated by the fact that activity of enzymes is adversely affected by high temperatures. As shown in Figure 2 and 3, the reaction rate increases with temperature to a maximum level, then abruptly declines with further increase of temperature. Because this enzyme rapidly become denatured at temperatures above 40 °C in contrast to most of enzymes that denatured at temperatures above 40 ºC. peroxidase is very resistant at high temperature such 45 ºC, so that peroxidase remain 40 % of its activity at 50 ºC. These results showed that potential of activation for peroxidase is very high. It has been noted that heat stability of the enzyme may be related to ripeness of the fruit and molecular forms of the enzyme, and in some cases it is also dependent on pH (Zhou and Feng, 1991). Rapid decrease in activity in high temperature after optimum temperature might be due to involvement of disulfide bond in the active site or in three dimensional conformation of the enzyme. There are several other reports that describe high temperature stability of peroxidase from other sources to the same temperature range. The drop in percentage residual activity at high temperatures can actually be due to the unfolding of the tertiary structure of the enzyme to form the secondary structure (Lowrenco et al., 1990).

Acknowledgment
This work was supported in part by the University of Payame Noor, and was done in exploratory laboratory of biochemistry in payame noor of saghez (Kurdistan).