Pertanika 9(2), 219 - 224 (1986)

Polyphenoloxidase from Starfruit (Averrhoa carambola, L.) I

TENGKU ADNIN BIN TENGKU ADNAN, M.A. AUGUSTIN and HASANAH MOHD. GHAZALI Department ofFood Sc~'ence, Faculty ofFood Science and Technology, Un'iversiti Pertanian Malaysz"a, 43400 Serdang, Selangor, Malaysia.

Key words: Polyphenoloxidase; starfruit; catechol. ABSTRAK

Polifenoloksidase (PPO) telah diasingkan dar'ipada belz"mbing besi (Averrhoa carambola, L.) dan ditulenkan melalui pemendakan aseton dan dialisis. pH optimum untuk pengoksidaan katekol yang d'imangkinkan oleh PPO yang dz"tulenkan dengan aseton adalah 7.2. Kajian penyahaktifan haba menunjukkan bahawa enzim PPO adalah labil terhadap haba. Hayat-separa untuk penyahaktifan PPO pada 70°C dan 75°C mas'ing-masing ialah 4.6 m'in dan 3.1 min. Didapatijuga bahawa malar 1 Michaelis (K dan V untuk katekol dengan PPO masing-masing ialah O. 061M dan 20 U ml- • J max ABSTRACT

Polyphenolox'idase (PPO) was z50lated from starfru'it (Averrhoa carambola, L.) and purif'ied by acetone preC£pz"tat'ion and d'ialysis. The optimum pH for the aCl}tone purif'ied PPO catalysed ox'idat£on of catechol was found to be pH 7.2 Heat 'inact£vat'ion stud'ies showed that the enzyme was heat-labile. Haif-Hvesfor PPO 'inact'ivat'ion at 70°C and 75°C were found to be 4.6 m'in and 3.1 m'in respect'ively. The M'ichaelis constant (K j and V Jor catechol with PPO was O. 061M and 20 U ml- 1 respect'ively.

INTRODUCTION polyphenoloxidase. It has generally been found that PPO is a relatively difficult enzyme to Starfruit (Averrhoa carambola, L.) is con­ extract and purify (Mayer and Hare!, 1979). sidered to be under the oxalidaceae or oxalis Although there appears to be a wealth of infor­ family. Like most other fruits and vegetables, mation on the subject, the overall picture of the starfruit discolours during storage or when it polyphenoloxidase is not complete. is cut and processed. The discolouration leading to browning is a consequence of non-enzymic as This is because (i) PPO acts on a great well as enzymic reactions involving polyphenolic number· of substrates and catalyses more than .compounds. The specific enzymes which take one reaction (ii) PPO can occur in multiple part in browning reactions have been referred to forms in individual species (iii) the physiological generally as polyphenoloxidases (Mayer and role of the enzyme is not understood and (iv) it Harel, 1979; Vamos-Vigyazo, 1981). (Enzyme has a complex role in food processing (Vamos­ number 1.14.18.1 with the systematic name of Vigyazo, 1981). monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; Anon, 1973). It is also clear that the characteristics of A considerable amount of work has been PPO vary with the type of fruit. There are dif­ done on the isolation and characterization of ferences between PPO from different cultivars

1 Presented in part as a Poster at the 11th Malaysian Biochemical Society Conference, 9th-10th September, 1985. TENGKU ADNIN BIN TENGKU ADNAN, M.A. AUGUSTIN AND HASANAH MOHD. GHAZALI and also differences in PPO isolated at different enzymatic phenol oxidation and polymerization stages of maturity of fruits and vegetables. This during extraction. This may cause irreversible has become evident from reports on PPO from inactivation of the enzyme and a resultant low peaches (Wong et ai., 1971; Luh and Phit­ activity of the extracted enzyme (Vamos­ hakpol, 1972; Flurkey and Jen, 1978), banana Vigyazo, 1981). In this work, PVP which is a (Galeazzi and Sgarbieri, 1981; Galeazzi et ai., phenol scavenger was added to the extraction 1981; Jayaraman et ai., 1982), cherries buffer during the isolation step. This minimizes (Benjamin and Montgomery, 1973), guava enzyme denaturation by phenol oxidation (Augustin et ai., 1985), pears (H~m and products (Stelzig et ai., 1972). Triton-X was Montgomery, 1978; Montgomery and \Petro­ incorporated into the extraction medium pakis, 1980), avocado (Kahn, 1975; Kahn, 1977) because it functions as a solubilization agent. and mango (Park et ai., 1980). The charac­ teristics of PPO from a large variety of fruits and Purification of the crude extract involved vegetables has been reviewed by Vamos-Vigyazo acetone precipitation and dialysis. All purifica­ (1981). tion steps were carried out at 4°C. The acetone precipitate was obtained by addition of 2 There has been no work reported on star­ volumes of cold acetone and recentrifugation at fruit PPO. An understanding of the enzyme will 15 min at 12 000 rpm (25 500 g) at 4°C. The help efforts in controlling the undesirable precipitate was reextracted with one volume of browning of the fruit and processed starfruit 0.2M phosphate buffer (pH 6.8). For the dialysis products. The present work involved the extrac­ step, the reextracted solution containing the tion and purification of PPO from starfruit and acetone-precipitated enzyme was put into a a study of its enzymic activity using catechol as a dialysis bag (Sigma Dialysis 'Sacks' 250-7U) and substrate. dialysed against 0.2M phosphate buffer (pH 6.8). The dialysis was carried out at 4°C for 48 hrs MATERIALS AND METHODS with four changes of dialysing media.

Materials PPO Assay Starfruits were obtained from the area around Universiti Pertanian Malaysia. The age Enzyme activity was determined by measur­ of the fruit was estimated at 1.5 months. The ing the increase in absorbance at 410 nm. The fruits had a yellowish-green colour. reaction mixture contained 1 ml 0.05M catechol, varying amounts of enzyme extract and Extraction and Purification ofPPO 0.2M phosphate buffer (pH 6.8) in a final volume of 5 m!. The temperature of the assay The method used was based on that of was 30°C. The rate of the reaction was calculat­ Galeazzi et ai., (1981). 100 g of longitudinally ed from the initial linear slopes of activity curves. sliced fruit was homogenized in a blender for 30 One unit (U) of PPO activity was defined as the sec with 100 ml of 0.2M phosphate buffer (pH amount of the enzyme that increased the absor­ 6.8) containing 1.5% polyvinylpyrrolidone bance by 0.001 min - I under the conditions of (PVP) and 0.5% Triton X-100. Homogenates the assay (Benjamin and Montgomery, 1973; were centrifuged at 12 000 rpm (25,500 g) for 15 Galeazzi et ai., 198.1). min at 4°C (Du Pont RC-5B Centrifuge). The enzyme remained in the supernatant (crude The pH profile of the enzyme was deter­ extract). mined in th range of pH 5.0 - pH 8.0 using 0.2M phosphate buffer and the conditions of Previous reports have shown that one of the the assay described above. Specific activity major difficulties encountered during prepara­ (U mg - I) was plotted against pH to get the pH tion of a soluble PPO is that of preventing optimum.

220 PERTANIKA VOL. 9 NO.2, 1986 POLYPHENOLOXIDASE FROM STARFRUIT (AVERRHOA CARAMBOLA, L.)

The thermal denaturation of the enzyme ed in a 9.5 and 22 fold increase in the specific (acetone-precipitated extract) was obtained by activity. adding 3 ml of the enzyme extract in test-tubes and heating at 70 oe, 75°C and 80 0 e respec­ Acetone precipitation has often been used tively. After various heating intervals, 0.5 ml in extraction and purification steps for PPO. samples were withdrawn and immediately cooled Flurkey and Jen (1978) found that activities of in ice-water (Park et at., 1980; Wissemann and peach PPO were greatly enhanced by acetone Lee, 1981). It was then assayed for PPO activity powder preparation. It was suggested that the with 0.05M catechol as the substrate at pH 6.8 20-fold increase in PPO activities in extracts of under the conditions of the assay described acetone powder over that of fresh extracts of above. peach PPO was due to the presence of inhibitory substances in fresh extracts or possibly because of

Determinatz'on ofK m and V max aggregation of PPO isoenzyme forms during acetone powder preparations (Flurkey and Jen, The Michaelis constant (K m) and the 1978). The possibility of these factors coming maximum velocity (V ) were determined by into play in the starfruit PPO preparation can­ varying the catechol c~~centrations of the re­ not be excluded. The further increase in specific action mixture over the range 0.02M - 0.10M activity after dialysis also suggests the removal of catechol. The temperature and pH of the re­ other inhibitory low-molecular weight sub­ action mixture was 30 0 e and pH 6.8 respec­ stances from the enzyme extract. tively. The data was plotted as 1/ activity against 1/ catechol concentration. pH Profile

Protein Determination Initial experiments on the aCtiVity of the crude extract showed that the pH optimum in Protein concentration of the extracts was the range pH 5.0 - 8.0 was pH 7.3. The pH determined using the Lowry method as given by profile of the acetone purified extract is given in Layne (1957). Bovine serum albumin was used as Figure 1. The pH optimum of this extract was the standard. found to be pH 7.2. It has been stated that the purity of the enzyme extract can affect the pH RESULTS AND DISCUSSION optimum tStelzig et al., 1972) but this was not evident in the case of the crude extract and a Isolation and Purification ofPPO acetone purified extract from starfruit. The pH optimum of most PPO's studied have been The results of the·extraction and purifica­ shown to be in the range pH 5.0 -7.0. It has also tion of starfruit PPO are given in Table 1. The been found that pH optimum of the enzyme can crude extract had low specific activity. Purifica­ be influenced by the type of phenolic substrate tion by acetone precipitation and dialysis result- being oxidized. Differences in pH optima can

TABLE 1 Extraction and purification of starfruit PPO

Stage Protein Specific activity Purification mg ml- l Umg- l fold

Crude extract 14.2 2.6 ± 1.0 1 Acetone precipitate 0.65 24.7 ± 3.1 9.5 Dialysed enzyme 0.19 56.7 ± 1.1 22

PERTANIKA VOL. 9 NO.2, 1986 221 TENGKU ADNIN BIN TENGKU ADNAN, M.A. AUGUSTIN AND HASANAH MOHD. GHAZALI

the enzyme and/or conformational changes 25 /1',,1 , which result in shifts in its pH optimum (Mayer I\ and Harel, 1979). I \ / " \ \ I \ / \ / \ , \ ,I ;, Thermal lnactz'vatz'on ofStarfruz't PPO ,: \ , I , \ I \ Fz'gure 2 shows the results of the heat inacti­ 20 I \ : \ , \ vation of the acetone purified PPO at 70°C and 75°C. As expected, the rate of enzyme inactiva­ i \ tion increased with increasing temperature. I -I b() Heat inactivation at 80°C for 2 min resulted in a E \ 95 loss of enzyme activity. 2- 15 t/ % ;>< \ ... \ I ;; \ \ 1= \ u« 100 u \'\, ii: 0 10 we; '-'" ""'" .. , 50 ...., ,, ,, '., 5 , . ,, '."" . , , 7.0 7.2 7.4 2 4 6 8 10 pH

Fig. 1: pH profile of the acetone puTtfied extract TIME (MIN) from Starfruit. Fz'g. 2: Thermal Inactivation of Starfruit PPO (---- 70°C; e 75°C).

also occur when PPO is obtained from different The linearity of the plots of log aCtiVIty cultivars of the fruit (Mayer and Harel, 1979). against time showed that heat inactivation followed first order kinetics. The half lives' for A pH optimum of 7 has been found for a heat de~aturationat 70°C and 75°C ~ere found number of other PPO preparations. Among to be 4.6 min and 3.1 min respectively. Studies some of them with a pH optimum of 7 for PPO into the heat inactivation of PPO from mango activity are PPO from certain varieties of apple, peel and mango (Park et al., 1980; Katwa et al., pear, sweet cherry, mandarin, banana, mango, 1983), grape (Wissemann and Lee, 1981, Lee et mushroom and green chilly (Vamos-Vigyazo, al., 1983), pear (Halim and Montgomery, 1978) 1981). However, it should be borne in mind that and cherries (Benjamin and Montgomery, 1973) pH optimum as well as ~he relationship between have shown first order kinetics of denaturation. activity and pH can differ according to genera, cultivar and substrates (Vamos-Vigyazo, 1981). Changes in pH profile of PPO activity can some­ A comparison of the heat lability ofstarfruit times be accompanied by activation of the PPO with PPO from other sources show that enzyme in the neutral to alkaline region of the . starfruit PPO is more susceptible to heat pH curve. This is due to partial denaturation of denaturation than PPO extracted from De

222 PERTANIKA VOL. 9 NO.2, 1986 POLYPHENOLOXIDASE FROM STARFRUIT (AVERRHOA CARAMBOLA, L.)

Chaunac grapes, d'Anjou pears, mango and ~ mango peel. Half lives for heat inactivation of -, De Chaunac grapes were about 3.5 min and 5.4 2.20 min at 75°C and 70°C respectively (Lee et al., ..>­ ~ 1983). PPO from d'Anjou pears were even more :> ;:: resistant to heat denaturation as shown by half u.10 lives of 6.3 min and 11.7 min at 75°C and 70°C ~ respectively (Halim and Montgomery, 1978). Katwa et al. (1983) found that PPO from mango peel lost 50% of its activity by exposure to 75°C 40 20 0 20 40 for 16 min. [CATE CHOlf' (~-')

Fz"g. 3: Double-reczprocal plot - 1/Activity against The results on the heat lability of PPO are l/(catechol). in. line with the observation that PPO's are not extremely heat stable enzymes. Short exposures to temperatures of 70°C to 90°C are generally sufficient to cause ir_reversible inactivation of the CONCLUSION enzyme (Vamos-Vigyazo, 1981). The paper described the characteristics of a partially purified PPO enzyme extracted from K and V Values m max starfruit. In any work on PPO, it should be noted that there may be limitations because the The effect of varying catechol concentra­ characteristics can be affected by maturity of the tion is shown by the double reciprocal plot in fruit and the method of extraction and purifi­ Figure 3. The values of K and V were m x cation. Furthermore, there is a possibility of the 0.061M and 20 U ml -lrespecti~elyat pH 6.8. occurrence of isoenzymes and interconversion between different forms of the enzyme during extraction and purification. Luh and Phithakpol (1972) found that K m for cling peach PPO was 0.015M while the K m for Elberta freestone peach was 0.12M with ACKNOWLEDGEMENTS catechol at pH 6.2 (Reyes and Luh, 1960). Mango, eggplant and banana PPO with catechol This research was funded by Universiti as substrate gave K m values of O.0082M (Katwa, Pertanian Malaysia. 1983), 0.007M (Roudsari et al., 1981) and 0.0018M (Galeazzi and Sgarbieri, 1981) respec­ REFERENCES tively. These differences in K m values suggest that the affinity of PPO towards a given sub­ ANON., Enzyme Nomenc1q.ture, Elsevier, Amsterdam, strate can vary within wide limits. The pH of the 1973. enzyme assay also affects the ultilizability of the AUGUSTIN, M.A., HASANAH MOHD. GHAZALI and substrate (Vamos-Vigyazo, 1981). Smaller K HASIMAH HASHIM (1985): Polyphenoloxidase m values are obtained for a stronger binding of the from guava (Psidium guajava, L). j. Sci. Fd. Agric. 36: 12§9' - 1265. substrate to the enzyme. It has been suggested BENJAMIN. N.D. and MONTGOMERY, M.W. (1973): that differences in K m values for PPO mfty be Polyphenol oxidase of Royal Ann cherries: Purifi­ due in part to steric factors arising from dif­ cation and Characterization. j. Food Sci. 38: ferences in protein structure. A recent review 'by 799 - 806. Vamos-Vigyazo (1981) lists K values for PPO m FLURKEY, W.H. and JEN. J.J. (1978): Peroxidase and from different fruits and cultivars which further PPO activites in developing peaches. j. Food Sci. illustrates this point. 1826 - 1831.

PERTANIKA VOL. 9 NO.2, 1986 223 TENGKU ADNIN BIN TENGKU ADNAN, M.A. AUGUSTIN AND HASANAH MOHD. GHAZALI

GALEAZZI, M.A.M. and SGARBIERI, V.C. (1981): LUH, B.S. and PHITHAKPOL, B. (1972): Characteristics Substrate specificity and inhibition of polypheno­ of polyphenoloxidase related to browning in cling loxidase (PPO) from a dwarf variety of banana peaches.I Food Sci. 37: 264 - 268. (Musa cavendishz'i, L.). Food Sci. 46: 1404­ I MAYER. A.M. and HAREL, R.E. (1979): Polyphenol 1406. oxidases in plants. Phytochem. 18: 193 - 215. GALEAZZI. M.A.M., SGARBIERI. V.C. and CONSTAN­ MONTGOMERY. M.W. and PETROPAKIS, H.J. (1980): TINIDES, S.M. (1981): Isolation, purification and Inactivation of Bartlett pear polyphenoloxidase physiochemical characterization of polypheno­ with heat in the presence of ascorbic acid.I Food loxidase (PPO) from a dwarf variety of banana Sci. 45: 1090 - 1091. (Musa cavendishii, L.). I Food Sci. 46: 150 -155. PARK, Y.K., SATO, H.H., ALMEIDA, T.D. and MORETTI, R.H. (1980): Polyphenol oxidase of HALIM. D.H. and MONTGOMERY, M.W. (1978): Poly- mango (Mangifera indica, var. Haden). I Food phenoloxidase of d'Anjou pears (Pyrus Sci. 45: 1619 - 1621. communis, L.).I Food Sci. 43: 603 - 608. REYES, P. and LUH, B.S. (1960): Characteristics of JAYARAMAN. K.S., RAMANA]A, M.N., DHAKNE, Y.S. browning enzymes in Fay Elberta freestone and VIjAYARAGHAVAN, P.K. (1982): Enzymatic peaches: Food Technol. 14: 570. browning in some varieties as related to' PPO activity and other endogenous factors. Fd. Sci. I ROUDSARI, M.H., SIGNORET, A. and CROUZET, J. & Tech. 19: 181 - 185. (1981): Eggplant polyphenoloxidase: purifica­ KAHN. V. (1975): Polyphenoloxidase activity and tion' characterization and properties. Food browning of three avocado varieties. I Sci. Fd. Chem. 7: 227 - 235. Agric. 26: 1319-1324. STELZIG, D.A., AKHTAR, S. and RIBEIRO, S. (1972): KAHN, V. (1977): Some biochemical properties of Catechol oxidase of red delicious apples. Phyto­ polyphenoloxidase from two avocado vari.eties chern. 11: 535 - 539. differing in their browning rates. Food SCl. 42: I VAMOS-VIGYAZO, L. (1981): Polyphenol oxidase and 38 -43. peroxidase in fruits and vegetables. CRC Rev. KATWA, L.C., RAMAKRISHNA, M. and RAGHAVENDRA Food Sci. and Nutrition 15: 49 - 127. RAO. M.R. (1983): Purification and properties of WISSEMANN, K.W. and LEE, C.Y. (1981): Characteri­ PPO from mango peel (Mangifera indica, var zation of PPO from Ravat 51 and Niagara Raspuri).I 6: 217 - 228. Food Biochem. grapes.I Food Sci. 46: 506 - 508. LAYNE, E. (1957): Spectrophotometric and turbi­ WONG, T.C., LUH, B.S. and WHITAKER, J.R. (1971): metric methods for measuring proteins. Methods Isolation and characterization of polyphenol 3: 447 - 454. ofEnzymology oxidase isoenzymes of Clingstone peach. Plant LEE. C.Y., SMITH. N.L. and PENNESI, A.P. (1983): Physiol. 48: 19-23. Polyphenoloxidase from De Chaunac grapes. I Sci. Fd. Agric. 34: 987 - 991. (Received 16 November, 1985)

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