Polyphenol oxidase (PPO) is a common copper-containing enzyme which is widely distributed in nature. In the presence of oxygen it catalyses two reactions: the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones (van Gelder, Flurkey, and Wichers, 1997; RodrÄ±´guez-Lo´pez et al., 2001). This enzyme plays an important role in many plant metabolic processes. The oxidation of phenol compounds by PPO are polymerised to form dark-coloured compounds responsible for browning in fruits and vegetables. This is regarded as an undesirable sensory attribute of the produce decreasing its commercial and nutritive values. For this reason PPO received increasing attention. Thus the properties of the enzyme have been reviewed by several worker as Vamos-Vigyazo (1981), Mayer (1987), Zawistowski, Biliaderis,and Eskin (1991), and Yoruk and Marshall (2003). PPO enzyme has been isolated and characterised from produce of many commercially important plants such as broad beans (Ganesa, Fox, and Flurkey, 1992), plums (Siddiq, Sinha, and Cash, 1992), potatoes (Chen et al., 1992; Marri, Frazzoli, Hochkoeppler, and Poggi, 2003), lettuce (Heimdal et al., 1994; Gawlik-Dziki, Zlotek, and Swieca, 2008), apples (Espin, Morales, Varon, Tudela, and Garcia-Carnovas, 1995), pears (Hwang, Yoon, and Kim, 1996), coffee (Mazzafera and Robinson, 2000), peppermint (Kavrayan and Aydemir, 2001), avocado (Gomez-Lopez, 2002), and broccoli (Gawlik-Dziki, Szymanowska, and Baraniak, 2007).
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Few studies dealt with the purification and characterisation of PPO from coloured petals. The present investigation covers the purification of PPO, effects of pH and temperature on PPO activity and stability, effects of selected chemical compounds as inhibitors of PPO activity and substrate concentration and specificity of PPO from petals of Hibiscus rosa-sinensis L. The results add to current information needed to elucidate the properties of the PPO responsible for the browning of fruits and vegetables during storage thus shortening their shelf-life.
Materials and methods
Fresh red flowers of Hibiscus rosa-sinensis grown in the Botanic Garden of MUST were hand picked from the plant in a summer morning, placed in polyethylene bags, and stored at ...?°C until further extraction and analysis. (Lyophilisation)!
Enzyme extraction and partial purification
The enzyme was extracted following the method described by ??? and ???(19??).
?? grams of petals were homogenized in ?? mL of ??M phosphate buffer pH ?? containing ?? and ?? and extracted with the aid of a magnetic stirrer for ?? h. The crude extract samples were centrifuged at ?????g for ??min at the temperature of ??°C. Solid ammonium sulphate (NH4)2SO4 was added to the supernatant to obtain ??% saturation. After an hour, the precipitated proteins were separated by centrifugation at ?????g for ?? min. The precipitate was re-dissolved in small volume of ??M phosphate buffer (pH ??) and dialyzed at ?? °C against the same buffer for ?? h with ?? changes of the buffer during dialysis. The protein content and PPO activity were monitored in the dialysed solution spectrophotometrically.
Polyphenol oxidase activity assay
The polyphenol oxidase activity was determined by measuring the absorbance at 420 nm using a spectrophotometer (Pharmatech, Model UV-1700) as described by ??? and ????? (19??). To determine the best concentration of enzyme preparation corresponding to the highest enzyme activity, the activity was assayed in 3 mL of reaction mixture consisting of 0.5 mL substrate (0.02 M 4-methylpyrocatechol or 0.02 catechol) and different concentrations of the enzyme preparation (0.025, 0.05, 0.1, 0.2, and 0.5 mL enzyme preparation) and completed to 3mL with the phosphate buffer (pH 6.8). The blank consisted of 3.0 mL phosphate buffer (pH 6.8). Two controls were prepared: the cuvette of the first control contained 2.7 mL buffer solution and 0.5 mL substrate, whereas the second control cuvette contained 2.8 mL buffer and 0.2 mL enzyme preparation. Absorbance values of these controls were subtracted from that of the sample. The increase in absorbance was linear with time for the first 120 s. The initial rate was calculated from the slope of the absorbance-time curve. The results were expressed as absorbance increment/min. An increase in absorbance of 0.001 min-1 was taken as one unit of enzyme activity (Ho, 1999). An enzyme preparation of 0.1mL showed the highest activity using 4-methylpyrocatechol as substrate.
Substrate concentration and specificity of PPO
An attempt was made to select the best substrate and its optimum concentration corresponding to the highest enzyme activity. The PPO activity was determined using different concentrations of catechol (0.01, 0.02, 0.04, 0.08, and 0.16mM) and different concentrations of 4-methylpyrocatechol (0.01, 0.02, 0.04, 0.08, and 0.16mM). The activity was assayed in 3 mL of reaction mixture consisting of 2.6 mL phosphate buffer (pH 6.8), 0.3 mL substrate and 0.1mL enzyme preparation. The highest enzyme activity corresponded to the concentration of 0.04M of 4-methylpyrocatechol which was used for subsequent experiments.
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Protein content was estimated by the protein assay method of Lowry et al (1951). Bovine serum albumin was used as a reference protein to prepare standards for the assay.
Effect of pH on enzyme activity
The effect of pH on PPO activity was determined under standard laboratory conditions using 0.1 mL enzyme preparation, 0.3 of 0.04M 4-methypyrolcatechol and completed to 3mL of 0.1 M sodium acetate buffer (pH 3.0,4.0, 4.5, 5.0, and 5.5) or 0.1 M sodium phosphate buffer (pH 4.5, 6.5, 7.0, and 7.4). The optimal pH was that value corresponding to the highest enzyme activity.
Effect of inhibitors on PPO activity
The effects of four potential inhibitors (ascorbic acid, ???????, copper sulphate, EDTA (ethylenediaminetetraacetic acid sodium salt) on PPO activity were evaluated using 0.04m 4-methypyrolcatechol as substrate. The reaction mixture was incubated at 25° C (standard laboratory conditions) and the change in absorbance was measured spectrophotometri cally at 420 nm. A control test containing the same concentration of enzyme but without the inhibitor was run. Results were expressed as ?????????????.