Purification and properties of polyphenoloxidase of mango peel (Mangifera indica)

J. Biosci., Vol. 4, Number 1, March 1982, pp. 69-78. Printed in India. Purification and properties of polyphenoloxidase of mango peel (Mangifera indica) T. N. PRABHA and M. V. PATWARDHAN Discipline of Fruit and Vegetable Technology, Central Food Technological Research Institute. Mysore 570 013 MS received 6 November 1981: revised 9 January 1982 Abstract. Polyphenoloxidase from mango (Mangifera indica) peel was purified to homogeneity by ammonium sulphate fractionation, chromatography on DEAE-Sephadex and gel filtration of Sephadex G-200. The enzyme had an apparent molecular weight of 136,000. Its ph and temperature optimum were 5.4 and 50 C, respectively. The enzyme possessed catecholase activity and was specific to o-dihydroxy phenols. The enzyme also exhibited peroxidase activity. Some non-oxidizable phenolic compounds inhibited the enzyme competitively. High inhibitory effects were also shown by some metal chelators and reducing agents, Mango peel polyphenol oxidase when immobilized onto DEAE Sephadex showed slightly higher K m for catechol and lower ph and temperature optima. Keywords. Mango (Mangifera indica) peel ; polyphenoloxidase; peroxidase; immobilized enzyme. Introduction Polyphenoloxidase (EC 1.14.18.1) which plays a major role in the browning of plant tissues has been purified from a number of fruit tissues and their properties studied (Mayer and Harel, 1979; Jen and Flurkey, 1979; Jen et al., 1980; Shin and Maier, 1980; Wissemann and Lee, 1980). The enzyme from many of the sources is shown to exist in multiple forms (Mayer and Harel, 1979). It is known to be a copper containing enzyme. Polyphenol oxidase from Mangifera indica (mango peel, Badami variety) was partially purified and some of its properties namely, ph optimum, substrate specificity, and presence of copper in the enzyme were demonstrated in an earlier report (Venkiah and Patwardhan, 1977). This paper presents an improved method of purification of the enzyme from the same source leading to a homogeneous preparation thus enabling us to study many more of its properties in detail. Attempts are being made to immobilize polyphenol oxidase for its possible use in instant tea manufacture (Prabha et al., 1981). The present communication also reports the properties of the purified mango peel polyphenol oxidase immobilized onto DEAE-Sephadex as compared to the soluble enzyme. Abbreviation used: PVP, polyvinyl pyrrolidone. 69

70 Polyphenoloxidase of mango Materials and methods Fully mature mango fruits (Badami variety) harvested from local gardens were allowed to ripen at room temperature. When they were edible ripe, the peel portion of the fruits was collected for enzyme isolation. DEAE-Sephadex A-50 and Sephadex G-200 were from Pharmacia Fine Chemicals, Uppsala, Sweden; standard proteins and polyphenols were from Sigma Chemical Co., St. Louis, Missouri, USA. Enzyme isolation Mango peel was blended in 1 mm potassium phosphate buffer, ph 7.4 containing 1% soluble polyvinyl pyrrolidone (PVP) and 0.1% ascorbic acid and acetone powder of this crude extract was prepared. Acetone powder was extracted for 1 h with about 10 volumes of 10 mm potassium phosphate buffer, ph 7.4 containing 1% insoluble PVP and 0.25% ascorbic acid. The viscous slurry thus obtained was clarified by treating with immobilized fungal pectinase for 2 h with intermittent stirring. Fungal (Aspergillus carbonareus) pectinase was immobilized by entrapping in Polyacrylamide gel according to Chibata et al. (1976). The block gel containing entrapped pectinase was rubbed through a 40 mesh sieve and the small gel beads obtained were washed repeatedly with water to remove unbound pectinase. The polyphenoloxidase extract thus clarified was filtered through a cheese cloth and centrifuged at 10,000 g for 10 min to get a clear solution. Enzyme purification All purification steps were carried out at 4 C. The enzyme was precipated from the crude extract at 70-80% ammonium sulphate saturation and suspended in a minimum volume of 0.05M phosphate buffer, ph 7.4 and dialyzed against 0.01Μ phosphate buffer (3L) overnight. The above enzyme was placed on a DEAE-Sephadex column (3.5 13 cm). Potassium phosphate buffer, ph 7.4, 0.025M, 0.05M, 0.075M and 0.1M (flow rate: 12 ml/h) 100 ml were passed through the column. The enzyme activity was detected in the fraction eluted with 0.1 Μ phosphate buffer. The eluate from DEAE sephadex column was concentrated by lyophilization to about 2 ml and further purified by gel filtration. Gel filtration of the enzyme was carried out on a Sephadex G-200 column (1.4 150 cm; flow rate: 5ml/h) with 0.1M potassium phosphate buffer, ph 7.4. The enzyme fractions were pooled and used in the study. For molecular weight determination, the enzyme was rechromatographed (Whitaker, 1963) on the same column previously calibrated with blue dextran and other standard proteins. The proteins were monitored by their absorbance at 280 nm. The enzyme was monitored by assaying for activity. Enzyme assay Polyphenol oxidase activity was measured spectrophotometrically by the method described by Wong et al. (1971) using 10 mm catechol as substrate. One unit of the enzyme activity was taken as increase in absorbance at 420 nm of 0.01 per min per mg protein. In the case of immobilized preparation, about 2g of the material

Prabha and Patwardhan 71 (drained) was incubated with 10 ml of 20 mm catechol for 60 min at 25 C. The absorbance of the supernatant solution was measured at 420 nm. The reading was recorded every 15 min. Here, the activity unit was taken as increase in absorbance at 420 nm of 0.01 per 30 min per mg protein. To determine the enzyme activity in the presence of inhibitors, the enzyme was preincubated for about 15 min with these compounds. Peroxidase activity was measured by the method described by Vance and Sherwood (1976). Protein was estimated according to Lowry et al. (1951). Electrophoresis Disc gel electrophoresis of the enzyme preparations (sample containing ca. 100 μg protein) was carried out on 7.5% Polyacrylamide gels using Tris-glycine buffer, ph 8.3 according to Davis (1964). The gels were stained with 5% amidoblack solution for 30 min and destained with 7% acetic acid to develop protein bands. The polyphenoloxidase bands were detected by dipping the gels in 10 mm substrate solutions containing 0.05% p-phenylenediamine (Montgomery and Sgarbieri, 1975). Peroxidase bands were developed with hydrogen peroxide and o-dianisidine (Shannon et al, 1966). Immobilization by adsorption of polyphenol oxidase on to DEAE Sephadex Adsorption of the enzyme on DEAE-Sephadex A-50 was achieved by suspending about 20 ml of the enzyme solution with 5 g (wet weight) of pre-conditioned support at ph 7.4 using 0.1M phosphate buffer with constant stirring for 2 h as described by Park and Toma (1975). This was then washed free of unbound enzyme. Quantity of protein held on the support was calculated by subtracting the protein present in the combined washings of the immobilized support from the protein taken for immobilization. Results Table 1 gives the results of purification of polyphenol oxidase. More than 90% of the enzyme protein was precipitated at 70-85% ammonium sulphate saturation. Table 1. Purification of mango peel polyphenoloxidase.

72 Polyphenoloxidase of mango The enzyme fraction after ion exchange chromatography with DEAE Sephadex and gel filtration on Sephadex G-200 was nearly 100-fold purer. Polyacrylamide gel electrophoresis of this fraction revealed a single protein band. This enzyme protein corresponded to the activity band when stained for activity (figure 1) and Figure 1. Polyacrylamide gel electrophoretic pattern of different fractions obtained during purification of mango peel polyphenoloxidase. (a) 70-85% ammonium sulphate fraction; (b) DEAE Sephadex fraction; (c) Sephadex G-200 fraction. (a), (b) and (c) are stained for polyphenol oxidase activity; (d) same as (e) but stained for proteins; (e) same as (c) but stained for peroxidase activity. this corresponded with one of the activity bands in the crude extract. The same protein band also possessed peroxidase activity when stained for peroxidase. The four isoenzymes of polyphenoloxidase present in the crude extracts exhibited different substrate specificities, as revealed by the differential appearance of activity bands upon incubation with catechol, dopa or dopamine, gallic acid, chlorogenic acid and caffeic acid. The molecular weight of the purified polyphenol oxidase isoenzyme was found to be 136,000 by gel filtration (figure 2). The purified enzyme had optimal activity at ph 5.4 and at 50 C. Figure 3 represents temperature stability curve of the purified polyphenoloxidase. Significant loss in activity was noticed at temperature 70 C and above.

Prabha and Patwardhan 73 Figure 2. Molecular weight determination of mango Peel Polyphenoloxidase by gel filtration on Sephadex G-200 Figure 3. Temperature stability curve for mango peel polyphenoloxidase. 15 min, (ο); 30 min, ( ); and 60 min, (Δ) The substrate specificity of the purified enzyme is shown in table 2. Catechin and epicatechin had an oxidation rate which was about 75% higher than that of catechol, whereas other substrates exhibited nearly 50% lower activity than catechol. The enzyme was inactive towards hydroquinone, p-cournaric acid and tyrosine. K m values of the enzyme for various phenolics derived from Lineweaver- Burk plots were 3.49 mm, catechol; 1.9 mm, catechin; 2.0 mm, epicatechnin; 8.9 mm, gallic acid; 6.9 mm, dopamine and 12.4 mm, caffeic acid. Inhibition of polyphenoloxidase by various classes of inhibitors is shown in table 3. Some of the non-oxidizable phenolics, viz., hydroquinone, pyrogallol and α- naphthol inhibited the enzyme activity by about 80% at 1 mm concentration. The inhibition of the enzyme by these phenolics except p-coumaric acid was competitive

74 Polyphenoloxidase of mango Table 2. Specificity of the purified mango peel polyphenol oxidase. * Phenolics were added at l0mm concentration. Activities are expressed relative to catechol, which is taken as 100. Not determined. Table 3. Inhibition of mango peel polyphenol oxidase by different classes of inhibitors * Compounds like benzoic acid, salicylic acid, resorcinol, cinnamic acid, p-chlorophenol, m-, o-and p-cresols and diacetyl. Not determined.

Prabha and Patwardhan 75 as evidenced by the Lineweaver-Burk plots (figure 4). The K m values of the enzyme for catechol in presence of. hydroquinone, pyrogallol, α-naphthol and p-coumaric acid increased from 3.49 to 20.16, 17.64,15.12 and 5.7 mm respectively (figure 4) As indicated in table 3, other phenolics did not inhibit the enzyme even at 1 mm concentration. Figure 4. Lineweaver-Burk plots of mango peel polyphenoloxidase for catechol in presence of phenolic inhibitors (10 mm). Hydroquinone (ο); pyrogallol ( ); α-naphthol (Δ); p-coumaric acid ( ); control ( ). Metal chelating compounds, namely 3,4-dichlorophenyl serine, sodium azide, sodium diethyldithiocarbamate and phenylthiourea caused more than 75% inhibition at 0.1 mm concentration. 3,4-Dichlorophenyl serine was highly inhibitive even at 1 mm level. EDTA did not show an inhibition. The reducing agents-glutathione, cysteine, potassium metabisulphite and ascorbic acid completely inhibited the enzyme activity at 0.1 mm level, glutathione being the most potent. Other compounds like calcium chloride, borate and PVP at 1 mm level showed very little effect on mango peel polyphenoloxidase. The effect of some of the selected inhibitors of polyphenoloxidase on peroxidase activity of the same enzyme protein is given in table 4. p-coumaric acid and ferulic acid at 1 mm level showed a high activation of peroxidase activity while inhibiting the polyphenoloxidase activity of the protein. 3,4-Dichlorophenyl serine (copper chelator and a potent inhibitor of polyphenol oxidase) had no effect on peroxidase activity. On the other hand, 1, 10-phenanthroline hydrate (a specific iron chelator) brought about total inhibition of peroxidase activity even at 10 μμ level and this metal chelator had no effect on polyphenoloxidase activity.

76 Polyphenoloxidase of mango Table 4. Effect of some inhibitors of polyphenol oxidase on peroxidase activity of the purified enzyme proteins. Values are expressed as per cent increase or decrease over the control. (+): activation; ( ) : inhibition; 0: no effect. The enzyme exhibited good storage stability. The enzyme stored in ph 7.4 buffer at room temperature for 72 h or at 5 C for 2 months retained 95% of the original activity. But at ph 5.5, the enzyme lost about 50% of the activity under similar conditions. Freeze drying, freezing and thawing did not affect the activity of the enzyme. Acetone powders which were stored at 15 C retained 90% of the activity upto 4 months. A storage period of 6, 8 and 10 months decreased the activity to 60, 55 and 30% respectively. The isoenzyme pattern of mango peel polyphenoloxidase from acetone powers stored for more than 8 months showed diffused bands of the enzyme with a poor resolution. Properties of the immobilized polyphenoloxidase The purified enzyme immobilized onto DEAE Sephadex showed linear activity between 10 and 70 min, as against 0-4 min for the soluble enzyme. The insoluble enzyme exhibited an optimum ph of 5.0 and an optimum temperature of 44 C. The immobilized enzyme exposed for 60 min to 50,60,70 and 80 C retained 90,80, 22 and 0% activity respectively. The K m of the bound enzyme for catechol was 7.2 mm which was 3.49 for the soluble enzyme. The immobilized enzyme showed higher activity towards catechin and epicatechin as compared to catechol (3 and 2.9-fold respectively) and showed much lower activity towards dopamine, gallic acid, chlorogenic acid and caffeic acid than catechol (0.4, 0.42, 0.4 and 0.38 fold respectivley). The specific activity of the native enzyme decreased upon immobilization by about 18%. The immobilized mango peel polyphenoloxidase retained more than 90% of its activity even at the end of six months storage at 4 C. Discussion In this study, mango peel polyphenoloxidase has been obtained in an electrophoretically homogeneous form. This purified enzyme protein corresponds to isoenzyme-2 of the four isoenzyme bands originally present in the crude extract.

Prabha and Patwardhan 77 The only report on the existence of isoenzyme forms for mango peel polyphenol oxidase has been that of Park et al. (1980) who reported two isoenzymes reacting with catechol in Haden variety of mango. The four isoenzymes of mango peel noticed in our study exhibited differences in their reactivity towards various substrates, of which the isoenzyme-2, was reactive to all o-dihydroxyphenols and was the most prominent one. Venkiah and Patwardhan (1977) purified mango peel (Badami variety) polyphenoloxidase to 50-fold with 4% recovery by ion-exchange chromatography and demonstrated the presence of copper, the substrate specificity and the ph optimum for the partially purified preparation. The polyphenoloxidase enzyme from mango peel has been purified to homogenity with about 11% recovery in the present investigation. The purified mango peel enzyme has peroxidase activity, similar to the polyphenoloxidase from other sources (Shannon et al., 1966; Bayse and Morrison, 1971; Hideakishinishi, 1975). 1, 10-Phenanthroline hydrate, a specific iron chelator inhibits peroxidase activity of the mango peel enzyme, but not polyphenoloxidase activity. Similarly, 3, 4-dichlorophenyl serine, a specific copper chelator inhibited polyphenoloxidase activity but not peroxidase activity. Thus, it could be inferred that the purified enzyme from mango peel is a metalloprotein containing both copper and iron. Further, copper is not involved in peroxidase activity of the protein and iron is not involved in polyphenoloxidase activity of the enzyme protein. Immobilization of mango peel polyphenol oxidase by adsorption onto DEAE Sephadex has resulted in slight shifts in ph and temperature optimum towards the lower side. The K m for catechol has increased from 3.49 to 7.2 mm, associated with fall in specific activity. Similar loss in specific activity upon immobilization has been reported for aminoacylase adsorbed onto DEAE-Sephadex (Tosa et al. 1966). Loss in enzyme activity upon immobilization has been attributed to change in enzyme conformation imposing rigidity and hindered accessibility of the enzyme to substrate (Zaborsky, 1974). Immobilization of mango peel polyphenoloxidase did not bring about drastic changes in specificity of the enzyme towards phenolic substrates. Immobilized preparation of the enzyme suspended in buffer and kept at 4 C has shown good storage stability for 6 months. Generally, immobilization brings about modifications in the properties of the enzyme as has been reviewed by Zaborsky (1974). During extraction of ripe mango peel with buffer, a lot of pectin was co-extracted resulting in a highly viscous non-filterable mass, which prevented a clean separation of the aqueous phase containing the enzyme. To overcome this problem, pectinase was made use of in our work. Immobilized pectinase (entrapped in Polyacrylamide gel) for clarifying the mango peel extract was most advantageous and therefore was adopted. Immobilized pectinase was preferred to soluble enzyme as the former could be removed easily after use so that additional protein contamination is avoided.

78 Polyphenoloxidase of mango References Bayse, G. S. and Morrison, M. (1971) Biochem. Biophys. Acta, 244, 17. Chibata, I., Tosa, T. and Sato, T. (1976) Appl. Microbiol., 27, 878. Davis, Β. J. (1964) Ann. Ν. Υ, Acad. Sci., 121, 404. Hideakishinshi, (1975) Phytochemistry, 14, 1255. Jen, J. J. and Flurky, W. H. (1979) Hort. Sci., 14, 516. Jen, J. J., Seo, A. and Flurky, W. H., (1980) J. Food Sci., 45, 60. Lowry, Ο. Η., Rosebrough, Ν. J., Farr, Α. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Mayer, A. M. and Harel, E. (1979) Phytochemistry, 18, 193. Montogomery, M. W. and Sagarbieri, V. C. (1975) Phytochemistry, 14, 1245. Park, Y. K. and Toma, M. (1975) J. Food Sci., 40, 1112. Park, Y. K., Sato, H. H., Almeida, T. D. and Moreiti, H. H. (1980), J.Food Sci., 45, 1619, Prabha, T. N., Ramakrishna, M., Patwardhan, M. V. and Raghavendra Rao, M. R., (1982) Indian J. Biochem. Biophys. (in Press). Prabha, Τ. Ν. (1981) Changes in polyphenols and related enzymes during fruit ripening Ph. D. thesis submitted to the University of Mysore. Shannon, L., Kay, E. and Lew, J. (1966) J.Biol. Chem., 241, 383. Shin, M. and Maier, V. P. (1980)7. Agric. Food Chem., 28, 891. Tosa, T., Mori, T., Fuse, N. and Chibata, I. (1966) Enzymologia, 31, 214. Vance, C. P, and Sherwood, R. T. (1976) Plant Physiol., 57, 916. Venkiah, B. and Patwardhan, Μ. V. (1977) J. Food Sci. Technol., 14,173. Whitaker, J. R. (1963) Anal. Chem., 35, 1950. Wissemann, Κ. W. and Lee, C. Y. (1980) J. Chromatog., 192, 232. Wong, T. C, Luh, B. S. and Whitaker, J. R. (1971) Plant Physiol., 48, 19. Zaborsky, O. (1974) in Immobilized enzymes, (Cleveland: CRC Press).