v . _ .uv ..‘_--. .. ., . . . . V . . .. ‘ ...: .. . .' .... A . xv, .... L . . ‘\.\ .. ‘ .. . . ... v V. . .A. .31 ‘., .. . , L A. lllllllllllllllllIllllllllllllllllllllllllllllllIlllllllllllll 31293 00908 9883 This is to certify that the dissertation entitled Characterization of polyphenol oxidase from Stanley plums and a study of its involvement in anthocyanin loss in plum juice presented by Muhammad Siddiq has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in Date August 5, 1993 MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan state University PLACE IN RETURN BOX to tern ove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ~ JU .. ‘ 331—11200; 1023 01 1:5 U 8 MOS v N x 7’3 N l MSU Is An Affirmative Action/Equal Opportunity Institution ckianG-pd ,, 7 \ CHARACTERIZATION OF POLY PI-IENOL OXIDASE FROM STANLEY PLUMS AND A STUDY OF ITS INVOLVEMENT IN ANT HOCY ANIN LOSS IN PLUM JUICE By Muhammad Siddiq A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1 993 ABSTRACT CHARACTERIZATION OF POLYPHENOL OXIDASE FROM STANLEY PLUMS AND A STUDY OF ITS INVOLVEMENT IN ANTHOCYANIN LOSS IN PLUM JUICE By Muhammad Siddiq Ten plum (Prunus domestica L.) cultivars were investigated for polyphenol oxidase (PPO) activity. Stanley plums, which showed highest PPO activity and are of commercial significance in Michigan, were selected for characterization and purification of this enzyme and its involvement in anthocyanin degradation in plum juice. The. activity of crude enzyme was 3.5 times greater in the flesh than in the skin of the fruit. The enzyme showed a Km of 20mM of catechol and a Vmax of 6.529 AA42onm/min x 10'1. Its pH and temperature optima were 6.0 and 20°C, respectively. Below pH 4.0 the enzyme lost most of its activity. Among the substrates, 4—methylcatechol followed by catechol, dopamine, pyrogallol and caffeic acid were readily oxidized by this enzyme. It was most stable at -20°C and retained about 80% of its activity at this temperature even after 1 year storage. The plum PPO was strongly inhibited by ascorbic acid, D-isoascorbic acid, L-cysteine, sodium diethyldithiocarbamic acid, sodium metabisulfite and thiourea. All of the benzoic and cinnamic acid series compounds proved to be poor inhibitors of plum PPO. Heating at 65 and 75°C for 30 and 5 minutes, respectively, rendered this enzyme completely inactive. Muhammad Siddiq Plum juice anthocyanins were relatively thermo-stable as a loss of only 16% was observed when the juice was heated for 70 minutes at 65°C. Loss of anthocyanins in the juice was directly related to the concentration of added PPO. When monitored for anthocyanin degradation over a 24 hour period, L- cysteine and sodium metabisulfite proved very effective in minimizing loss of these pigments in plum juice in the presence of added PPO. Neither ascorbic acid nor citric acid alone or in combination controlled anthocyanin loss. Crude PPO was purified 36-fold with about 22% yield through successive purification steps involving ammonium sulfate fractionation, dialysis, ion- exchange chromatography on DEAE-cellulose and gel-exclusion chromatography on Sephadex G-100. Both chromatographic steps resolved the enzyme into a predominantly single peak. Polyacrylamide gel electrophoresis (PAGE) of crude PPO showed 7 activity bands whereas purified PPO separated into 3 isoenzymes. The molecular weight of these 3 subunits of purified PPO on SDS-PAGE was estimated to be in the range of 45,000 to 66,000 daltons. IN THE NAME OF GOD THE COMPASSIONATE THE MERCIFUL Dedicated to cherish the loving memory of my father, Muhammad Ismail, to whom I stand in debt for my education and knowledge iv ACKNOWLEDGEMENT I am highly indebted to almighty Allah (God) Who enabled me to complete this manuscript. I wish to express my heart felt gratitude to my academic advisor, Dr. Jerry N. Cash, for his kind help, inspiring guidance, consistent encouragement and critical evaluation of the whole manuscript. Grateful appreciation is extended to Dr. Bruce Harte, Dr. Pericles Markakis, Dr. Theodore D. Wishnetsky and Dr. Michael V. Doyle for serving on my guidance committee, reviewing this manuscript and making many constructive suggestions and comments. I am extremely thankful to Dr. Denise M. Smith for allowing me to use equipment in her lab. For many helpful suggestions, technical assistance, critical evaluation and timely help in preparation of this dissertation, I owe a special debt of gratitude to Dr. Ninnal K. Sinha. Thanks are also due to all of my lab partners at Michigan State University, especially Tirza Hanum, who offered their invaluable help and encouragement throughout the course of present studies. Appreciation is extended to Michigan Agricultural Experiment Station, Michigan Food and Crop Bioprocessing Center and Michigan Plum Advisory Board for their suport of this research. Finally and for many reasons, I owe the greatest debt to my loving wife -- Asma, sweet daughters -- Raafia and Farihah, son Omair, mother, sister, brother, in- laws, relatives and friends, for their help, interest and concern which enabled me to reach this stage. TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ....................................................................................................... ix INTRODUCTION .................................................................................. 1 REVIEW OF LITERATURE ..... ' ................................................................................. 3 Nomenclature .................................................................................................... 3 Reactions of PFC .............................................................................................. 4 Occurence of PFC in nature ............................................................................ 7 Latency of PFC ................................................................................................. 9 Substrates of PFC ............................................................................................. 9 Phenol-protein complexation and interactions ................................ , ........... 11 Extraction methods of PPO ............................................................................. 15 Assay of PPO activity ...................................................................................... 16 Properties of PPO .............................................................................................. l7 Purification of PFC ..................................... _ ...................................................... 21 Isoenzymes of PFC .......................................................................................... 22 PPO action on anthocyanins .......................................................................... 23 Thermal inactivation of PFC ........................................................................... 24 Inhibition of PFC .............................................................................................. 25 MATERIALS AND METHODS .................... 29 MATERIALS .......................................................................................................... 29 Plum Samples ............................................................................................ 29 METHODS .............................................................................................................. 29 I. CHARACTERIZATION OF PLUM PPO .................................................... 29 Enzyme extraction ................................................................................... 29 Assay of enzyme activity ........................................................................ 30 Protein detenmnatlon ........................ 30 Enzyme kinetics .............................................................. ' ......................... 31 Substrate specificity ................................................................................ 31 pH optima. ................................................................................................. 32 Effect of temperature ............................................................................... 3 2 Storage stability ........................................................................................ 32 Effect of inhibitors ................................................................................... 33 Heat inactivation ....................................................................... . ............... 33 vi II. EFFECT OF PPO ON PLUM JUICE ANTHOCYANINS ............................ 34 Plum juice extraction ............................................................................... 34 Total phenolics and chlorogenic acid ............................... , ................... 34 Total anthocyanins in plum juice ........................................................... 35 Effect of PPO on plum juice anthocyanins ........................................... 36 Effect of heat treatment on plum juice anthocyanins ......................... 36 Effect of PPO inhibitors on plum juice anthocyanins ......................... 36 III. PURIFICATION OF PLUM PPO ................................................................... 36 Ammonium sulfate fractionation ............................................................ 37 Ion-exchange chromatography ............................................................. 37 Gel exclusion chromatography .............................................................. 3 9 Polyacrylamide gel electrophoresis ....................................................... 3 9 RESULTS AND DISCUSSION ............................................... 42 I. CHARACTERIZATION OF PLUM PPO ..................................................... ' 42 Substrate specificity ................................................................................ 42 Enzyme kinetics ....................................................................................... 45 pH optima. ................................................................................................. 46 Effect of temperature ............................................................................... 50 Storage stability ........................................................................................ 53 Efi'ect of inhibitors ................................................................................... 53 Heat inactivation ...................................................................................... 61 .II. EFFECT OF PPO ON PLUM ANTHOCY ANIN S ......................................... 63 Total phenolics and chlorogenic acid in plum cultivars ..................... 63 Eflect of heat treatment on plum juice anthocyanins ......................... 65 Efiect of PPO inhibitors on plum juice anthocyanins ......................... 65 III. PURIFICATION OF PLUM PPO ................................................................... 70 . DEAR-cellulose chromatography .......................................................... 72 Gel-exclusion chromatography on Sephadex G-100 ......................... 72 Polyacrylamide gel electrophorosis (PAGE) ........................................ 75 SUMMARY AND CONCLUSIONS .......................................................................... 78 SUMMARY .......................................................................................................... 78 CONCLUSIONS .................................................................................................. 80 BIBLIOGRAPHY ......................................................................................................... 82 APPENDICES ............................................................................................................... 99 vii Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. LIST OF TABLES Endogenous PPO substrates in foods .................................... 10 PPO activity in different plum cultivars ................................ 43 Substrate specificity .of plum PPO with different phenolic compounds ........................................................................ 44 Km and Vmax values of plum PPO with different substrates. 48 Total phenolics and chlorogenic acid content in different plum cultivars ................................................................... 64 Purification of plum PPO .................................................... 71 viii Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. LIST OF FIGURES Reactions catalyzed by polyphenol oxidase (PPO) ................ 6 Substrates of PPO present in foods .................................... 12 Polyphenol-protein complexation and precipitation ............. 13 pH optima of PPO from selected fruits ............................. 18 Temperature optima of PPO from selected fruits ............... 20 Lineweaver and Burk plot of plum PPO with catechol ........ 47 pH activity profile of plum PPO in sodium acetate and citrate phosphate buffer ................................................... 49 pH activity profile of plum PPO with different substrates... 51 Effect of temperature on plum PPO activity ...................... 52 Effect of different storage temperatures on plum PPO activity ........................................................................... 54 Effect of ascorbic acid and D-isoascorbic acid on plum PPO activity ................................................................... 55 Effect of benzoic acid series inhibitors on plum PPO activity ........................................................................... 57 Effect of cinnamic acid series inhibitors on plum PPO activity ................................................................... 58 Effect of L-cysteine, sodium diethyldithiocarbarnic acid, sodium metabisulfite and thiourea on plum PPO activity ..... 60 Heat inactivation of plum PPO at different temperatures ..... 62 ix Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Effect of heat treatment on plum juice anthocyanins at 65°C .............................................................................. 66 Effect of added PPO on the rate of anthocyanin loss in plum juice ..................................................................... 68 Effect of different PPO inhibitors at 0.25mM (A), 0.50mM (B) and 1.00mM (C) on anthocyanin loss in plum juice ....... 69 Ion-exchange chromatography of plum PPO on DEAE- cellulose ......................................................................... 73 Gel-exclusion chromatography of plum PPO on Sephadex G-100 ............................................................ 74 Polyacrylamide gel electrophoresis of plum PPO: SDS- PAGE of standard protein markers (Lane A) and purified PPO (Lane B), stained for protein; PAGE of crude (Lane C) and purified (Lane D) PPO stained for activity with catechol .................................................................. 76 APPENDICES Appendix 1. Standard curve for protein .......................................... 99 Appendix II. Standard curve for chlorogenic acid and total phenolics ................................................................... 100 INTRODUCTION Plum production is an important fruit industry both in Michigan and Pakistan. At present most of the fruit produced is consumed fresh, and the only processed forms of plums available in the market being plum paste, prunes and prune juice . Plum processed products have not been developed and marketed on a scale similar to some other fruits like apples, pears, apricots, etc. One of the possible reason for this has been a lack of research and development efforts in developing processed plum products, such as juice, concentrate and puree, for direct consumption or for use in some other finished food products. The post-harvest enzymic discoloration of plant tissue following cell damage in horticultural products is a serious problem. Polyphenol oxidase (PPO), which is the enzyme responsible for such browning, has been widely studied in many fruits and vegetables. As a result of PPO action the natural color of the product may be destroyed (in anthocyanin rich products) or impaired by the formation of dark brown pigments (in light colored products). Most of these changes occur during the early preparation stages of the fruit processing if proper measures to control PPO action are not taken promptly. In addition to discoloration of the product, its flavor and nutritive value are also negatively affected, which in turn results in poor acceptibility on the part of consumers. Most of the the work to prevent enzymic browning has "been aimed at four factors; substrate, enzyme(s), divalent cations and oxygen which are required for the enzyme catalyzed browning. Among compounds that have been shown to inhibit PPO activity, sulphur dioxide has been the most effective and has been 1 used in the food industry for many years. However, recent restrictions of sulfite usage coupled with consumers' concerns about its safety have generated the need for substitute PPO inhibitors. Besides chemical inhibitors, PPO may be inactivated by heat. However, this technique is often less desirable in anthocyanin rich products like cherries, grapes, plums, etc. because the high temperature necessary for PPO inactivation also causes anthocyanin degradation. The objectives of this study were to characterize and purify the PPO enzyme from plum fruit and study PPO induced changes in plum juice. This information will be useful in devising effective methods of inhibiting browning while at the same time minimizing anthocyanin pigment degradation in plum juice and other plum products. REVIEW oF LITERATURE ‘ Polyphenol oxidase (PPO) was detected in mushrooms for the first time over a century ago (Whitaker, 1972) and it occurs practically in all kinds of fruit with the exception of some citrus species. Ever since its discovery, PPO has been a subject of extensive research. Past research on this enzyme has been reviewed by various authors (Robb, 1984; Vamos-Vigyazo, 1981; Mayer and Harel, 1979; Mayer 1987). PPO belongs to the group of enzymes called oxidoreductases and is believed to be ubiquitous in the plant kingdom. Some trivial names of this enzyme, refering to the (principal) substrate(s) acted on, such as tyrosinase, cresolase, catecholase, catechol oxidase, phenolases, o-diphenol oxidase, are still in use in the literature (Vamos-Vigyazo, 1981). PPO is a copper containing enzyme that is responsible for the enzymatic browning or discoloration reactions in many fruit and vegetable products. Browning is undesirable not only because of the discoloration of the products but because the reaction produces off-flavor. PPO also causes a loss of characteristic color of anthocyanin containing fruit products (Co and Markakis, 1968). Synge (1975) reported that PPO impairs not only the sensory properties and, hence, the marketability of the product, but often lowers its nutritive value as well. Nomenclature Polyphenol oxidase (PPO) belongs to a group of enzymes called oxidoreductases. These enzymes oxidize diphenols in the presence of molecular 3 oxygen. The trivial name of this enzyme "polyphenol oxidase" has been applied in the early literature to denote two different enzymes (Anon., 1973). Previously these two enzymes were classified as catechol oxidase or o-diphenol oxygen oxidoreductase (EC 1.10.3.1) and laccase or p-diphenol oxygen oxidoreductase (EC 1.10.3.2). In 1973, with the abolition of subclass "10”, all the phenolases were re-classified as "monophenol monooxygenases" (EC 1.14.18.1) with catechol oxidase and laccase being combined as monophenol dihydroxy-L- phenylalanine oxygen oxidoreductase (Anon., 1973), commonly denoted in the literature as polyphenol oxidase. Mayer (1987) indicated that changes in international nomenclature have again resulted in monophenol monooxygenase (tyrosinase) being referred to as EC 1.14.18.1, diphenol oxidase (catechol oxidase, diphenol oxygen oxidoreductase) as EC 1.10.3.2 and laccase as 1.10.3.1. He further indicated that regardless of the grouping in the Intematidnal Union of Biochemists (IUB) classification, these enzymes are quite different in nature with respect to their substrate specificity. Reactions of PPO Catechol oxidase, now generally referred to as-PPO, catalyzes two types of specific oxidative reactions, (a) hydroxylation of monophenols (i.e. p-cresol) to o- diphenols (like 4-methylcatechol) referred to as cresolase activity, and (b) the further oxidation of o-diphenols (i.e. catechol) to o-quinones (like 0- benzoquinone), referred to as catecholase activity (Sanchez—Ferrer et al., 1988). A copper prosthetic group is involved in both reactions (Mason, 1955). Strothkamp and co-workers (1976) have demonstrated that copper atoms occur in pairs. A generalized scheme of basic reactions catalyzed by PPO, as illustrated by Whitaker (1972), is shown in Figure l. g The primary products of oxidative reaction catalyzed by PPO are 0- quinones. The o-quinones, (a) react with each other to form high molecular weight polymers, (b) form macromolecular complexes with amino acids or proteins, and, (c) oxidize compounds of lower oxidation-reduction potential. Reaction (c) is especially undesirable as the quinones formed by PPO, in addition to oxidizing compounds of lower oxidation-reduction potential, are themselve also reduced to dihydroxyphenols providing "fresh" substrate to the enzyme (Vamos-Vigyazo, 1981). Nelson and Dawson as early as 1944 postulated that a reducing agent was necessary to reduce the copper of the prosthetic group from cupric to the cuprous form to induce cresolase activity. Their views were also supported by Mason (1956). Thus, cresolase activity is characterized by an induction period which continues until sufficient amount of o-diphenols can be produced. They suggested that this induction period can be eliminated or reduced substantially by the addition of o-diphenols or other reducing agents. Catecholase activity is mostly responsible for enzymatic discoloration in fruits since a major portion of the phenolic compounds in the fruit are 0- diphenols. The o-quinones formed are highly reactive compounds and undergo oxidative polymerization to yield undesirable brown melanin pigments. The 0- quinones also react readily with amino acids and proteins to enhance this discoloration process (Mathew and Parpia, 1971). According to Walker (1975) the second enzyme, laccase, oxidizes o- and p- dihydroxy phenols but does not hydroxylate monophenols. This enzyme occurs O H O H OH O + 02 + B H2 ————9 0 Jr 8 + H2 0 CH5 0 H3 p-cresol 4-Methylcatechol A. Hydroxylation of monophenols to o-diphenols; cresolase activity OH 0 0 OH Catechol o-benzoquinone B. Oxidation of o-diphenols to o-benzoquinones; catecholase activity Figure 1. Reactions catalyzed by polyphenol oxidase (PPO). much less frequently than PPO in fruits and vegetables but has been reported in mushrooms (Brown, 1967; Turner et al., 1975), peaches (Mayer and Harel, 1968) and tomatoes (Filner et al., 1969). The current review and research will exclusively focus on catecholase activity of PFC, due to its predominant role in enzymatic discoloration in plums (Prunus domestica L. cv. Stanley; Siddiq et al., 1991). Occurence of PPO in nature PPO is widely distributed in plant kingdom. Its presence has also been reported in microoganisms especially in fungi and some animal organs (Brown, 1967). The black, brown, buff, and blue pigments found in feathers, hairs, eyes, insect cuticles, fruit, and seeds are usually melanins and are assumed to result from the action of tyrosinase. Thus it is not surprising that tyrosinase appears to be widely distributed both in the plant and animal kingdom. However, the enzyme has not been shown to be universal; it occurs relatively rarely in prokaryotes and is absent in a variety of higher plants such as Cucurbita and Brassica species (Robb, 1984). Tolbert (1973) reported that localization of PFC in plant cells depends on the species, age, and in fruits and vegetables on maturity. Craft (1966) observed that in case of potato tuber nearly all subcellular fractions were found to contain PPO, in amounts approximately proportional to protein contents. According to Stephen and Wood (1974), most of the activity in the stem of spinach beet was sedimentable and associated with plastid membranes and mitochondria. Harel et al. (1964) found that, in freshly harvested apples, the enzyme was observed to be localized in chloroplasts and mitochondria. They further indicated that PPO preparations obtained from these two particulate fractions differed slightly with respect to affinity towards different substrates. Vamos-Vigyazo (1981) reported that PPO distribution in different parts of fruits and vegetables may be considerably variable, and the ratio of particle- bound and soluble enzymes varies with maturity. Higher enzyme activity was found in the skin of grapes than in the flesh or sap. Its activity decreased during ripening, the decrease being mostly observed in the skir'1(Ivanov, 1966). Harel et a1. (1964) indicated that during ripening, the concentration of the particulate enzyme decreased with simultaneous appearance of soluble fraction. According to Olimpienko (1964) PPO content of plants might be affected also by agricultural techniques. He reported that activity levels in sugar beet leaves increased upon treating the soil with copper as a trace element. Sal'kova et al. (1977) reported that ethrel treatment of apple trees suppressed enzyme activity and FPO induced browning on cut surfaces of apples slices. PPO has been studied in a wide variety of fruits, such as apples (Satjawatcharaphong et al., 1983; Coseteng and Lee, 1987; Janovitz-Klapp et al., 1989 & 1990), cherimoya or "custard apple" (Martinez-Cayuela et al., 1988), peaches (Jen and Kahler, 1974; Flurkey and Jen, 1980), banana (Palmer, 1963; Kahn, 1985; Cano et al., 1990), grapes (Harel and Mayer, 1971; Cash et al., 1976; Nakamura et al., 1983; Sanchez-Ferrer et al., 1988; Valero et al., 1988 & 1991), guava (Augustin et al., 1985), lychee fruit (Huang et al., 1990), pears (Rivas and Whitaker, 1973; Smith and Montgomery, 1985; Zhou and Feng, 1991), mango (Park et al., 1980), olives ( Ben-Shalom et al., 1977; Sciancalepore, 1985), oranges (Bruemmer and Roe, 1970), plums (Arnold et al., 1992; Siddiq et al., 1992 a & b) and tea leaf (T akeo, 1966). Latency of PFC Existence of PPO to be present in latent form has been reported in apples ' (Harel et al., 1965), broad bean leaves (Kenton, 1957), sugar beet (Mayer, 1966), spinach (Tolbert, 1973; Sato and Hasegawa, 1976), grapes (Lerner et al., 1972; Harel et al., 1973). Sato and Hasegawa (1976) studied the latency of phenolase in spinach chloroplast and found out that isolated chloroplast of spinach leaves showed only a slight phenolase activity, which could be enhanced 4-9 fold with detergents like Triton X-100, Tween 80, sodium dodecyl sulfate, etc. They concluded that detergents effected both the activation of latent enzyme and its dissociation (release) from the membrane structure. Based on their results they concluded that the latency of spinach chloroplast phenolase is achieved in two ways; one, by firm binding of the enzyme with the membrane structure of the chloroplast and, two, by formation of an inactive complex from active protein and a low molecular weight substance. Robb (1984) suggested that one of the possible reasons that PPO is not active in vivo is that it is complexed with an inhibitor. He suggested the possibility that in vivo the enzyme can be rendered latent by the presence of sulfhydryl compounds, such as reduced glutathione (GSH). He'theorized that the membrane damage results in a more oxidizing environment, which favors oxidized glutathione and pigment formation, because the enzyme is not inhibited by the oxidized form of reduced glutathione. Substrates of PPO Schwimmer (1981) reviewed some of the substrates found in common foods (Table 1). - Chlorogenic acid and tyrosine are two the most commmonly 10 Table 1. Endogenous PPO substrates in Foods* Reported Substrates Food Apples Chlorogenic acid (flesh), o-catechin (peel) Banana 3,4-dihydroxyphenylethylamine (Dopamine) Cocoa epi-catechin Coffee beans Chlorogenic and caffeic acid Dates Caffeoyl shikimic acid Eggplant Caffeic, coumaric, cinnamic acid derivatives Fava beans Dihydroxyphenylalanine (DOPA) Lettuce Tyrosine Mushroom Tyrosine Olives Urushiol Potatoes Tyrosine, chlorogenic acid, flavonols Quinces Chlorogenic acid, catechin, flavonols, Sweet potatoes Tea leucoanthocyanidins Chlorogenic acid Flavonols, catechin, tannin *From Schwimmer (1981) ll occuring natural substrates of PPO in foods. Structures of some of these substrates are shown in Figure 2. Link and Walker (1933) reported that simple phenols such as catechol, which are commonly used in assaying phenolase activity, are usually not found in association with the enzyme although catechol has been reported to be present in the skin of the onion. Catechol, 4—methylcatechol, caffeic acid, chlorogenic acid, dopamine, pyrogallol, dihydroxyphenylalanine (DOPA), hydroqmnone and catechin are frequently used to assay catecholase activity of the PPO. Phenol, p-cresol and L- tyrosine are used to assay cresolase activity of the PPO. Moutounet and Mondies (1976) found chlorogenic acid, catechin, caffeic acid and DOPA to be naturally present substrates of PFC in plums (cv. d'Ente). Catechol which is absent in most plants continues to be the main substrate used in PPO assay studies (Cash et al., 1976; Flurkey and Jen, 1980; Amolds et al., 1991). Polyphenol-protein complexation and interactions Spencer et al. (1988) hypothesized that the association of polyphenols with proteins is principally a surface phenomenon. At a lower concentration of proteins the polyphenols associate at one or more sites on the protein surface, to give a mono-layer which is less hydrophillic than the protein (Figure 3a). This results in aggregation and precipatation. When the protein concentration is high a relatively hydrophobic surface layer is formed by complexation of the polyphenols onto the protein and by cross-linking of different protein molecules (Figure 3b). In such cases precipitation follows the above pattern. Based on this hypothesis, Spencer and co-workers further suggested that even simple phenols such as pyrogallol were also capable of precipitating proteins from solution if they CHchNHZCOOH 0H Tyrosine CH=CHCOOH H c H300 \TH HO o/Czo Scopotetin 12 CHZCHNHICOOH OH Dihydroxyphenylalanine (DOPA) it CH=CH-C—G C CH *5/64 {300“ I H " 0., .46" OH OH on Chlorogenic Acid 014. . on H0 0 / / ‘—‘OH \ \ 3 OH An Anthocyonidine Figure 2. Substrates of PFC present in foods. 13 (a) V\ F“. .i __, M Polyphenol H Low Prorein conc. (b) 1 Protein —7 ‘ \\\\\ dolyphenol x . '.‘\\“. \. \\\\\ _.‘ High PI'otein conc. (c) ‘ e . g Protein . 7 O O Phenol mono-layer -- excludes solvent hydrOphobic exterior Protein-phenol, protein-polyphenol precipitation Figure 3. Polyphenol-protein complexation and precipation. 14 can be maintained in solutions at concentrations sufficient to push the equilibrium in favor of the phenol-protein complex, as is illustrated in Figure 3c. White (1957) indicated that polyphenol-protein complexes can be dissociated with the addition of more proteins, hydrophobic solvents or hydrogen bond acceptors (such as acetones), urea, polyvinyl pyrrolidone (PVP), non-ionic detergents, etc. Furthermore, this complex formation is pH dependent. He stated that in cases where measurements have been possible the extent of complex formation rapidly declines when the pH of the medium is raised above 9.0. On the basis of all these observations White suggested that polyphenols interact with proteins by the formation of strong non-covalent bonds rather than by the action of ionic or covalent linkages. Loomis and Battaile (1966) reported that generally tannins interact with protein by multiple hydrogen bonds. This causes precipitation (Haslam, 1974) and also interferes with the protein digestion (Synge, 1975). Loomis (1974) classified the types of reactions resulting from the interaction of phenolic compounds with protein in a plant tissue into four major categories - hydrogen bonding, covalent coupling and ionic and hydrophobic interactions. Mason (1955) reviewed the work on covalent coupling reactions of quinones with proteins involving especially 1,4 additions. Pierpoint (1969) reported that the thiol group of cysteine to be particularly reactive. Among other reactive residues, alpha—amino group, especially that of peptides and proteins, and the imino group of proline were reported to be of significant importance. Synge (1975) reported that covalent coupling is the main cause of discoloration in plant tissues which may also reduce the nutritional quality of the proteins. 15 According to Loomis (1974), at high pHs, salt linkages may occur between basic amino acid residues and phenolic hydroxyl groups, due to relatively high pK value of phenolic hydroxyls. Formation of ionic bonds may occur at or below neutral pH values due to the negatively charged carboxyl group of certain phenyl-propanoid compounds. Hydrophobic bonding may also occur between the hydrophobic regions of proteins and the aromatic ring structure of the phenolic compounds. Extraction methods of PPO PPO from a variety of plant tissues has been extracted by preparation of acetone powders (Cash et al., 1976; Flurkey and Jen, 1980; Park et al., 1980; Lourenco et al., 1990; Zhou and Feng, 1991). Plant tissue is homogenized in a suitable buffer, usually citrate-phosphate buffer, filtered and precipitated by cold (-10 to -20°C) acetone. Acetone powder is suspended in the same buffer, centrifuged, and the supernatant used as crude PPO extract for characterization and purification. Special techniques have been developed to reduce or eliminate the interaction of PPO with other constituents of the plant cell during the extraction process. Phenolic scavangers, reducing agents and enzyme inhibitors have been used alone or in combination to improve PPO extraction and to retain its maximum activity. Phenolic scavangers are the most commonly used compounds for PPO extraction. These include insoluble polyvinyl pyrrolidone (PVP) (Loomis and Battaile, 1966), polyethylene glycol (Badran and Jones, .1965), Nylon®66 (McFarlane and Bayne, 1961), anion exchange resins (Lam and Shaw, 1970) and Amberlite® XAD resins (Loomis et al., 1979) are commonly used compounds in the extraction buffer.- l6 Insoluble PVP is the more frequently used phenolic scavanger. PVP, a high molecular weight polymer, has been reported to be ineffective at higher pHs due to the ionization of phenolic hydroxyl groups (Anderson and Sowers, 1968). They reported a pH of 3.5 as optimum for PVP-phenolic binding. Loomis (1974) also reported PVP to be most effective at neutral to acidic pHs. For best results, pH of the extraction buffer and that of the fruit from which PPO is being extracted must be taken into account for PVP to work. Anderson and Sower (1968) reported that PVP has a limited capicity to. adsorb chlorogenic acid. ' However, according to Loomis et al. (1979) PVP had high affinity for chlorogenic acid and decreased affinity for tyrosine at ph 6.5. Hsu et al. (1988b) reported that PVP was not very effective in removing phenolics from the crude enzyme homogenates. Assay of PPO activity Mayer et al. (1966) compared manometric, polarographic, chronometric and spectrophotometric methods for the determination of catechol oxidase activity, and recommended a polarigraphic method to be the most convenient and accurate. Walker (1975) recommended that to obtain accurate activity measurement with any of the methods it is important to monitor the initial rate, because the products of PPO reaction react with each other, unconsumed substrate, oxygen and proteins to alter further activity measurement. The spectrophotometric method is the most frequently used method to assay PPO activity in fruits and vegetables (Walker, 1962; Cash et al., 1976; Siddiq et al., 1991). This method measures increase in absorption, as a result of pigment produced by PPO action on phenolic substrates, at a specific wavelength. 17 Properties of PPO PPO from different plant tissues has been shown to have distinct properties such as pH and temperature optima, Km and Vmax. thermal stability, substrate specificity, etc. The PPO has been reported to be active over a wide range of pH between 4.0 and 7.0 (Figure 4). Thomas and Janave (1973) reported that PPO extracts from several sources were inactive below pH 4.0. However, Cash et al. (1976) observed that PPO from Concord grapes retained'50% of its activity even at pH as low as 3.4, the normal pH of grape juice. Moutounet and Mondies (1976) reported that d'Ente plums PPO had a pH optimum of 4.25 and even retained much of its activity at pH 3.8 which was the normal pH of the plum tissue. They reported that at pH 7.0 most of its activity was lost. Aylward and Haisman (1969) reported that the optimum pH for PPO activity greatly varies with the source of enzyme and with the substrate. Mihalyi et al. (1978) showed that not only the pH optimum, but the relationship between activity and pH over a wide range of pH values was found to differ with respect to genera, cultivars, and substrates. PPO preparations from the same fruit but at different stages of maturity have been found to differ in optimum pH of activity (Vamos-Vigyazo, 1981). According to Stelzig et al. (1972) the type of buffer and the purity of the enzyme may affect the optimum pH. They reported that particulate and soluble enzymes appeared to be essentially different in this respect. Most of the enzyme preparations have a single pH optima. According to Lanzarini et al. (1972) a second pH optimum was found in some cases resulting from insufficient purification of the enzyme. The temperature optimum of PPO activity has been not researched as extensively as pH optimum. From the research reported, it is quite evident that Relative Actlvlty Relative Activity (°/o) l8 LO - 100' -—O— d‘AMou —0— Outlet! 3 '9 _ + B'oec ‘3 - 30- 5; i o .7 - ‘. co- 1% '6 " ‘5 .s - 9 40- d .4 - d C .3 - 20' 2 - .I 0 fi fi— — J I I I I I I I I I 3 4 S 6 7 8 2 3 4 S 6 7 DH p" Pears (Siddiq et al., 1992a) Grapes (Cash et al., 1976) 10g— 80" 60' § 40- :lm" g. r- E 80" 8 60- 20- < ' E < D o 2 20 1 ;1 , , I ' . ‘ u 0 2 4 6 3 10 c: 0 pH Mushrooms (McCord and Kilara, 1983) Dates (Sachde et al., 1989) Figure 4. pH optima of PPO‘from selected fruits. l9 temperature optimum of PPO enzyme basically depends on the same factors which are responsible for variations in pH optimum (Vamos-Vigyazo, 1981). Temperature optima of PPO from different sources has been reported usually in the range of 20-40°C. Mihalyi et al. (1978) studied temperature optima of PPO activity from different fruits, and reported that PPO activity from Red Haven peaches increased from 3 to 37°C and then declined upto 45°C. In case of apricots the PPO activity reachedmaximum at 25°C. For Jonathan and Starking cultivars of apples, the activity was observed to'be maximum at 30 and 25°C, respectively. In potatoes, PPO was found to have its maximum activity with catechol at 22°C whereas for pyrogallol a nearly linear increase in activity was observed from 15 to 35°C. Figure 5 shows temperature optima for maximum PPO activity from some selected fruits. For Concord grapes PPO, Cash et al. (1976) reported a temperature optimum in the range of 25-30°C. PPO activity with respect to temperature optimum for three pear cultivars has been shown to differ considerably (Siddiq et al., 1992a). Michaelis-Menten constant (Km) and maximum velocity (Vmax) are a measure of affinity of an enzyme towards a substrate and velocity of an enzymatic reaction (at 2Km), respectively. Review of literature shows Km for PPO from different sources ranges from 0.01 mM to 67 mM. Affinity of PPO for a certain substrate may vary substantially, even if isoenzymes of the same source are considered (Vamos-Vigyazo, 1981). Moutounet and Mondies (1976) reported 3 Km of 13 mM with catechol for d'Ente plum PPO. No relationship has been found between Km and Vmax values determined for different substrates with a given PPO enzyme (Vamos-Vigyazo and Gajzago, 1978). PPO from different plant sources utilizes different phenolic substrates with variable degree of action, i.e. it is substrate specific. It is evident that PPO from Rolatlvo Actlvlty 20 1.0 - .9 - ' —o-— d°AflIOU g B _ too —-O—- Bartlett 9 ' —I-— 00.: 0 7 _ g. . oo- g .6 ‘g .5 ’3' 60- Q: .4 o q .3 40- .2 L J _ oar ' I r u . ' - ‘ v - I I I I I I I 1 I 10 20 so 40 so so 70 10l$202530354045505560 0 Temperature ( C) Term. C Pears (Siddiq et al., 1992a) Grapes (Cash et al., 1976) § ”100' . E :1. Z 30- Eso- "' >~ . U z < 60- £60” a 2 ~ __ Lo- ‘0_ e E . < - 2 E 20 g 20. g o i; in :81 :13 53 cf 70 60 so on o . . . . . O". n m ‘0 1f é ‘ 70 Dates (Sachde et al., 1989) Mushrooms (McCord and Kilara, 1983) Figure 5. Temperature optima of PPO from selected fruits. 21 most sources studied shows activity towards o-diphenols. PPO from apples (Coseteng and Lee, 1987) and kiwifruit (Ong et al., 1992) have -shown to act on both mono- and di-phenols. On the other hand PPO from apples (Janovitz-Klapp et al., 1989), banana (Palmer, 1963), eggplant (Sakamura et al., 1965), mushrooms (Bouchiloux et al., 1963; Constantinides and Bedford, 1967), peaches (Jen and Kahler, 1974), pears (Tate et al., 1964), and tea leaf (Takeo, 1966) exclusively exhibited only o-diphenol activity‘only. Sakamura et al. (1965) isolated PPO from eggplant which utilized several o-diphenols, including anthocyanins, as substrate. Molecular weight of PPO from different plant sources has been reported to be in the range of 17,000 to 180,000 (Flurkey and Jen, 1980; Hutcheson, et al., 1980; Wissemann and Montgomery, 1985; Sachde et al., 1989; Soderhall and Soderhall, 1989). Harel and Mayer (1968) reported three different fractions for apple PPO with the molecular weight of 40,000 to 130,000. PPO from two date cultivars was shown to have a molecular weight of 17,500 and 17,000, respectively (Sachde et al., 1989). Jolley et al. (1969) reported four subunits of mushroom PPO with a molecular weight of 32,400 for each. Contrary to this, Strothkamp et al. (1976) showed that mushroom PPO consisted of two subunits, a heavy (H) and light (L), with molecular weights of 43,000 and 13,400, respectively. Purification of PPO ¥ Different techniques, such as precipitation, gel-exclusion, ion-exchange and hydrophobic chromatography and isoelectric focusing, have been employed to purify PPO from different sources with varying success. Precipitation with ammonium sulfate of different saturation, gel-exclusion chromatography on Sephadex G-100 or G200, and ion- exchange chromatography on anion 22 exchangers DEAE-cellulose or DEAR-Sephadex are most commonly used techniques to remove low molecular weight proteins or impurities (Vamos- Vigyazo, 1981). The order of above purification steps is interchangeable, with occasionally one or more steps applied repeatedly (Sato, 1962). Flurkey and Jen (1978) purified peach PPO by hydrophobic chromatography on Phenyl- Sepharose CL-4B. Isoenzymes of PPO Markert and Moller (1959) defined isoenzymes as multiple molecular forms of enzymatically active proteins, catalyzing the same reaction and occuring in the same species, but differing in certain physical, chemical, and kinetic properties. Some earlier researchers speculated that specific types of enzymatic activity may be associated with more than one protein catalyzing the same reaction but having different chemical, physical and kinetic properties (Wieland and Pfleiderer, 1957; Markert and Moller, 1959). Mallette and Dawson (1949) were the first to report the presence of different forms of mushroom tyrosinase. ‘ Constantinides and Bedford (1967) studied multiple forms of phenoloxidase system in mushrooms, potato and apples. The enzyme system was shown to exhibit the phenomenon of multiple forms, which were characteristic for each individual species and variety studied -- with substrate specificity to be the most evident property. They reported that each multiple form behaved as an individual entity upon repeated elution and electrophoresis. PPO isoenzymes have been studied in apples (Walker and Hulme, 1966; Janovitz-Klapp et al., 1989), banana (Cano et al., 1990); dates (Sachde et al., 1989), grapes (Cash et al., 1976; Lee et al., 1983), walnuts (Piffaut and Metche, 1991), peaches (Wong et al., 1971; Flurkey and Jen, 1980), pears (Rivas and 23 Whitaker, 1973; Halim and Montgomery, 1978; Smith and Montgomery, 1985; Zhou and Feng, 1991), strawberries (Wesche-Ebeling and Montgomery, 1990), and many other fruits and vegetables. Bouchilloux et al. (1963) pointed out that the possibility of artifacts resulting from the isolation and extraction procedures must be considered. PPO action on anthocyanins Gross (1987) reported that water soluble anthocyanin pigments, ranging in color from red to blue, are one of the major classes of flavonoids. Their basic nucleus consists of two aromatic rings linked together by a three-carbon unit. An anthocyanin pigment is composed of an aglycon (an anthocyanidin) esterified to one or more sugars -- glucose, rharnnose, galactose, xylose and arabinose. Francis (1985) reported that anthocyanins may also be "acylated" with one or more molecules of p—coumaric, caffeic, ferulic, malonic, vanillic or acetic acids esterified to the sugar molecules. Plum anthocyanins are located mainly in the skin of the fruit. They accumulate in the vacuoles of epidermal and sub—epidermal tissue (Gross, 1987). Anthocyanins identified in different plum cultivars are: cyanidin-3-rutoniside, peonidin-B-rutoniside, cyanidin-B-glucoside and cyanidin-3-sambubioside (Timberlake, 1980; Druetta et al., 1985). The color of anthocyanins have been shown to be pH dependent (Markakis, 1974; Timberlake, 1980). They appear to be red in acidic media, blue or purple in alkaline media and almost colorless at intermediate pH values. Anthocyanins have been used as colorants in Maraschino cherries (McLellan and Cash, 1979), artificial grape drinks (Palamidis and Markakis, 1975), cherry pie (Volpe, 1976), dry beverage mixes (Shewfelt and Ahmed (1977) and many other 24 products. Sweeny and Iacobucci (1983) reported that the use of anthocyanins as colorant was restricted to foods or beverages having a pH below 4.0, because above this pH anthocyanins are rapidly decolorized. Naturally occuring enzymatic systems capable of decolorizing anthocyanins are present in molds, leaves, vegetables and fruits (Pifferi and Cultera, 1974). PPO, in addition to its action on colorless phenolic substrates, also acts on colored anthocyanins to decolorize them (Schwimmer, 1981). Such oxidation, whether direct or indirect, can influence food quality as demonstrated by Cash et al. (1976) on involvement of PPO in color loss of Concord grapes. Sakamura et al. (1966) isolated a PPO from eggplant which specifically oxidized the anthocyanins typical of that fruit. Bayer and Wegmann (1957) and Proctor and Creasy (1969) reported that crude vegetable extracts require the presence of phenols for any appreciable degradation of anthocyanins. They concluded that PPO is the enzyme responsible for such action. Peng and Markakis (1963) reported that anthocyanins alone were a poor substrate for mushroom PPO, but were quickly decolorized with the addition of catechol. Oszmianski and Lee (1991) also reported that anthocyanins themselves are not substrates of PPO but they are easily oxidized in the presence of chlorogenic acid. The destruction of anthocyanins, in such case, is attributed to its reaction with the quinone formed by oxidation of an appropriate phenol substrate. Thermal inactivation of PPO According to Vamos-Vigyazo (1981) PPO is not among extremely heat stable enzymes. Short exposure of PPO, in the tissue or in the solution, to temperatures of 70-90°C is usually sufficient for partial or complete irreversible 25 destruction of its catalytic capability. Thermal inactivation of PPO, like pH and temperature optimum, substrate specificity and other characteristics, also depends on the source of enzyme. Hare] et al. (1965) found that apple PPO was rapidly inactivated at temperatures above 70°C. At this temperature half life of apple PPO was 12 minutes and it was reported to be completely inactive at 80°C. Dang and Yankov (1970) studied the heat inactivation of PPO in 22 cultivars of different stone fruits. PPO from peaches was found to be least and from plums to be most heat stable. PPO was more heat stable in unripe than in ripe fruits. Inhibition of PPO Inhibition of PPO and resulting discoloration can be achieved by employing one or combination of several approaches. These include: a) specific inactivation of the enzyme itself, b) elimination of the substrate (3) of PPO in the product, c) interaction of a chelating compound with the copper prosthetic group, and d) elimination of oxygen required for PPO reaction (Vamos—Vigyazo, 1981). Robb (1984) reported that PPO inhibitors could be divided into three categories: a) general chelating agents for copper, b) non-competitive inhibitors with respect to the phenolic substrates, and c) analogs of phenols. _S_u_l_fi_te_s_: Among compounds that have shown to inhibit PPO, sulfites have been one of the most effective and traditionally used PPO inhibitors in the food idustry (Joslyn and Ponting, 1951; Shannon and Pratt, 1967; Langdon, 1987; Santerre et al., 1988; Sapers et al., 1989). I LuValle (1952) explained that sulfi'tes act as reductant compound by reacting with the o-benzoquinones to form a colorless complex. Embs and Markakis (1965). reported that the formation of such colorless complex prevents 26 the condensation of o-quinones which form dark pigments and this process continues until either the sulfite or the enzyme is completely consumed. In addition to this widely accepted view of indirect inhibition of PPO action by sulfite, inhibition of PPO itself by the sulfite has also been reported. Sayavedra- Soto and Montgomery (1986) suggested that major mode of direct and irreversible inhibition of PPO by the sulfite was modification of the protein structure with the retention of its molecular unity. Use of sulfite preservatives in fresh fruits and vegetables was banned in 1986 by Food and Drug Administration (FDA) due to its involvement in adverse health reaction in some individuals (FDA, 1986). Langdon (1987) reported that these adverse health effects mainly occured in asthmatic individuals. Ascorbic Acid : Ascorbic acid and its derivatives have also been used extensively in the food industry to inhibit PPO induced discoloration in fruit and vegetable products. With the ban on sulfites, research on the use of ascorbic acid and its formulations, and erythorbic acid and their sodium salts as an effective alternative to sulfites has been intensified (Duxbury, 1986; Langdon, 1987; Hsu et al., 1988a; Santerre et al., 1988; Sapers et al., 1987; Sapers and Hicks, 1989). Ascorbic acid retards enzymatic browning by re-reducing the quinones (intermediates of enzymatic browning) back to original substrates (Schwimmer, 1981). However, ascorbic acid is itself oxidized in the process, thus requiring large amounts for inhibition of enzymatic browning. Although ascorbic acid and few other compounds have shown some promise as PPO inhibitors, their degree of effectiveness is not as good as sulfites due to their poor ability to penetrate cellular matrix of the product (Taylor et al., 1986). Another problem reported by Ponting and Joslyn (1948) is that ascorbic 27 acid is easily oxidized by the endogenous enzymes or by iron- or copper- catalyzed reactions. Seib and Liao (1987) reported that ascorbic acid-2-phosphate and ascorbic acid-2-triphosphate were stable against oxidation and release ascorbic acid when hydrolyzed by phosphatase. Sapers et al. (1989) reported that ascorbic acid-2- phosphate and ascorbic acid-2-triphosphate have shown promise as inhibitors of enzymatic browning on cut surfaces of raw apples. Other PPO Inhibitors: Citric acid is another commonly used PPO inhibitor (Langdon, 1987; Santerre et al., 1988). Langdon (1987) reported that citric acid has double inhibitory effect on the phenolase by lowering the pH of the media and by chelating the copper portion of certain phenolases. Dudley and Hotchkiss (1989) suggested that, due to restriction on the use of sulfites in some food products, cysteine may present an effective alternative for controlling PPO action. Cysteine has also shown to have no effect on flavor intensity in d'Anjou pear juice concentrate (Montgomery, 1983). Other inhibitors of PPO, reviewed by Sapers and Hicksi(1989), include reducing agents (2-mercaptobenzothiazole, 2-mercaptoethanol and thioglycolate), quinone couplers (sodium diethyldithiocarbarnate, glutathione and benzenesulphinic acid), chelating agents (cyanide, carbon monoxide and diethyldithio-carbamate) and aromatic acids (benzoic acid and cinnamic acid). Some of these compounds are toxic, and none have found commercial use as food additives. Martinez-Cayuela et al. (1988) studied the inhibitory effect of some organic acids (acetic, oxalic, maleic, malonic, fumaric, succinic and glutaric acids) and sugars (D(+)glucose, D(-)fructose, and sucrose) on cherimoya epicarp PPO. However, these compounds were not able to inhibit the enzyme at the 28 concentrations used (0.1-1.0 mM). Pifferi et al. (1974) reported that acetic, maleic, fumaric, succinic, and oxalic acid slightly inhibited the sweet cherry PPO at higher inhibitor concentration (5.0 mM). Valero et al. (1991) successfully used tropolone, which is structurally analogous to o-diphenolic substrates, for inhibition of catecholase activity of grape PPO. Oszmianski and Lee (1990) studied the use of honey for inhibition of polyphenolase in apple and grape slices and model system. Chen et al. (1991) studied the inhibitory effect of kojic acid on'some plant and crustacean polyphenol oxidases. Recently, Dawley and Flurkey (1993) used 4- hexylresorcinol for inhibition of mushroom tyrosinase. MATERIALS AND METHODS MATERIALS Plum samples Ten plum cultivars (Prunus domestica L. cv. Abundance, Au Roadside, Beauty, La Cresecent, Pipestone, Pobeda, Shiro, Stanley, Underwood and Wade) grown in Michigan were used in the initial part of this study to compare PPO activity and concentration of total phenolics and chlorogenic acid. In the latter part, only Stanley cultivar plums were used for detailed characterization of .PPO and its action on plum juice anthocyanins. The plum samples were kept frozen at -20°C until required for enzyme extraction or further processing. METHODS I. CHARACTERIZATION OF PPO Enzyme extraction Extraction of enzyme was carried out using a modification of the method of Cash et al. (1976). All extraction materials were maintained at low temperature (2-5°C) to reduce enzymatic activity during extraction. A representative sample of about 50 g of tissue from 4-5 uniform sized plums was blended in Waring blender with 100 ml of 5°C 0.1M Tris hydroxymethyl aminomethane (T rizma) buffer (pH 9.5) for 2 minutes. The slun'y was filtered through 8 layers of cheese cloth and the filtrate was precipitated by slowly adding 200 ml of ~20°C acetone 29 30 with gentle stirring. When precipitation was complete, the precipitate was collected by straining through 45 micron mesh nylon cloth. The precipitate was suspended in 50 ml of 5°C 0.1M sodium acetate, pH 7.0. Pectic substances were precipitated by the addition of 8.0 ml of 0.05M calcium chloride. The solution was centrifuged in a refrigerated centrifuge at 4400 x g for 10 minutes and the supernatant was used as crude enzyme extract. Assay of enzyme activity Enzyme activity for PPO was assayed in duplicate according to the method of Cash et al. (1976). The standard reaction mixture consisted of 3.4 ml of 0.1M sodium acetate buffer (pH 6.0); 0.4 ml of 0.3M catechol; 'and 0.2 ml of freshly prepared enzyme extract. A lambda Perkin Elmer Spectrophotometer equilibrated at 30°C with enzyme kinetics software package was used to monitor change in absorbance at 420 nm for 3 minutes for assay of PPO activity. The increase in absorbance was recorded every minute. One unit of enzyme activity was calculated from the slope of the curve which determined AA420nm/min due to the oxidation of catechol (i.e. one unit = change in absorbance of 0.001/min). Protein determination Determination of protein content in the plum PPO extract was carried out using a dye binding technique (Coomassie Brilliant Blue G25) and bovine serum albumin as standard according to the method of Bradford (1976). A shift caused in the absorption maximum of the protein from 465 to 595 nm, as a result of dye binding, was measured to determine protein content. 31 The dye was prepared by dissolving 100 mg. of Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. To this 100 ml 85% (W/V) phosphoric acid were added and the resulting solution diluted to one liter with distilled water and then filtered through a milipore filter (0.47mm filter paper)./ A standard bovine serum albumin (BSA) solution was prepared by dissolving 100 mg dehydrated, moisture free BSA in 100 ml 0.15M NaCl. For protein measurement, amounts containing 10-100 pg of BSA (0.01 - 0.10 ml standard solution) were used. The volume of the standard protein solution was adjusted to 0.1 ml with the enzyme extraction buffer. Five ml of dye were added and the contents mixed well before measuring absorbance after 5 minutes at 596 nm against a blank made up of 5 ml dye and 0.1 ml extraction buffer. The standard curve of bovine serum albumin which is shown in Appendix I, was used to calculate the protein content in different enzyme preparations. Enzyme kinetics Michaelis-Menten constant (Km) and maximum velocity (Vmax), with catechol, 4-methylcatechol, chlorogenic acid and caffeic acid were determined. Substrate concentrations of 0.01-1.0M, except for chlorogenic and caffeic acid (0.01 - 0.5M), were used in the standard reaction mixture and PPO activity assayed. Data were plotted as l/activity against 1/ substrate concentration and Km and Vmax calculated according to the method of Lineweaver and Burk (1934). Substrate specificity Different commercial grade substrates, with mono-, di- or tri- hydroxy configuration, Were used to study plum PPO specificity at concentrations of 32 0.03M or 0.003M depending upon their solubility. Substrates with poor solubility were dissolved in 50% ethanol. Results are reported relative to PPO activity with catechol as 100%. pH optima Enzyme activities were determined with 0.3M catechol in 0.1M sodium acetate (pH 4.1-8.1) and 0.1M citrate-phosphate (pH 2.6-8.4). Enzyme extract was incubated with the buffer for 10 minutes at room temperature and substrate added before measuring absorbance. The optimum pH determined with sodium acetate buffer in this experiment was used in all subsequent characterization work. The pH optimum observed with catechol was also compared with pH optima determined with caffeic acid and 4—methylcatechol in 0.1M sodium acetate buffer (pH 3.5-8.0). Effect of temperature The activity of plum PPO at temperatures ranging from 710 to 70°C was determined. The standard reaction mixture in 10 ml test tubes without substrate was heated to the appropriate temperature in a water bath. After equilibration of the reaction mixture at the pre-deterermined temperature the contents were removed from the water bath, cooled in an ice bath for one minute before assaying PPO activity, as described above. Storage stability Storage stability of crude plum PPO was determined at temperatures of -20, 2 and 22°C for 16 weeks (pH 6.0). The activity was determined every week for first 4 weeks, then at 6, 8, 12 and 16 weeks. PPO samples stored at -20°C were 33 kept in separate vials for each observation to avoid repeated freeze-thaw effect on the enzyme. Effect of inhibitors Commercial grade PPO inhibitors from ascorbic acid series (ascorbic acid and D-isoascorbic acid), benzoic acid series (benzoic acid, vanillic acid and syringic acid), cinnamic acid series (t-cinnamic acid, p-coumaric acid and ferulic acid) as well as L-cysteine, sodium diethyldithiocarbamic acid, sodium metabisulfite and thiourea were used to study their effectiveness in inhibiting plum PPO (all these inhibitors were from Sigma Corp., St. Lious, MO) . A 0.1 ml aliquot of each inhibitor was added to the standard reaction mixture without the substrate to give a final concentration of 0.05, 0.25, 0.50, 0.75 or 1.00mM of the inhibitor. Before starting the reaction substrate was added and enzyme activity determined. The results are expressed as percent PPO inhibition. Some inhibitors with poor solubility were dissolved in 50% ethanol solution. Heat inactivation Heat inactivation pattern of plum PPO was determined in duplicate over a predetermined range of temperatures. Aliqouts of crude PPO in 0.1M sodium acetate buffer, were heated at temperatures of 45, 55, 65 and 75°C for 30 minutes. Residual PPO activity was determined at each temperature at 5, 10, 20 and 30 minutes of heating and results reported as % residual activity.- Heat inactivation was determined at pH 6.0. 34 II. EFFECT OF PPO ON PLUM JUICE ANTHOCYANINS Plum juice extraction Plum juice production was carried out according to the method of Arnold (1992). Forty five kg of Stanley plums were removed from -20°C and allowed to thaw overnight at 5°C. Debris (i.e. stems, leaves, shrivelled fruit) were removed. The plums were heated to 65°C and macerated in double jacketed stainless steel kettles. The macerated plums were cooled to 49°C and a commercial grade pectinase was added (1.0 g pectinase per 4.5 kg crushed fruit). After holding 6 hours at room temperature the crushed fruits were pressed to obtain juice using a rack and cloth press. The yield of plum juice was about 60%. The soluble solids content (14°B) and pH (3.9) of the juice were determined using an Abbe-3L refractomater (Bausch & Lomb Optical Co.) and a Corning 610A pH meter, respectively. The juice was stored at -20°C until required for further studies. Total phenolics and chlorogenic acid Total phenolics and chlorogenic acid in plums were determined by the method of Coseteng and Lee (1987). A 50 g sample of plums was homogenized in Waring blender with 100 ml 80% ethanol for 2 minutes. The homogenate was boiled for 5 minutes under the hood. The extract was first filtered through nylon cloth and then through Whatrnan #4 filter paper under vacuum. The residue was mixed with an additional 100 ml 80% ethanol and boiled for 10 minutes to re- extract the phenolics. The extracts were combined and made to a final volume of 250 ml. This extract was used for the determination of total phenolics and chlorogenic acid. For determination of chlorogenic acid 2 ml 5% sodium molybdate solution in 50% ethanol was added to 10 ml of the diluted alcohol extract. After mixing, a 35 10’ ml aliquot of the above solution was mixed with 2 ml 50% ethanol and absorbance was read at 370 nm. The concentration of chlorogenic was determined from a standard curve shown in Appendix II (10-60 fig chlorogenic acid/ml). The results were expressed as pg chlorogenic acid/gram of plums. To determine total phenolics, the alcohol extract was diluted in order to obtain an absorbance reading within the range of the standard (10-60 fig chlorogenic acid/ml). One ml of the diluted alcohol extract was added to 10 ml distilled water. Two ml Folin and Ciocalteau Phenol Reagent were added. The sample was mixed and after 5 minutes, 2 ml of saturated sodium carbonate solution were added. After one hour absorbance was read at 640 nm. The concentration of total phenolics was calculated from the standard curve (Appendix II). Total anthocyanins in plum juice The methods of Cash et al. (1976) were used to determine anthocyanin concentration and degradation spectrophotometrically at 535 nm. Frozen plum juice was thawed at room temperature for 45 minutes. Sample volume (9 ml) consisted of one part plum juice to two parts 0.025M citrate buffer. The diluted samples (pH 4.5) were kept in a 25°C water bath to maintain constant temperature. Total anthocyanins were extracted by mixing 1 ml of diluted sample with 9 ml of 95% ethanol :1.5N HCl in an 85:15 ratio (Skalski and Sistrunk, 1973). These samples were allowed to stand at room temperature for one hour before reading the absorbance at 535 nm. Changes in anthocyanin pigments were followed hourly for the first 8 hours and then at 16 and 24 hours. All the determinations were done in duplicate. 36 Effect of PPO on plum juice anthocyanins The PPO enzyme, which was extracted as described earlier, was added to the juice samples at a rate equal to 1.0, 5.0 and 10.0% activity of the plums (i.e. PPO from 1, 5 and 10 g plum tissue was added to 95 ml of diluted juice). Changes in anthocyanin pigments were monitored for 24 hours. Effect of heat treatment on plum juice anthocyanins Aliquots of diluted plum juice were placed in 10 ml glass test tubes and heated to 65°C (pasteurization temperature) in a water bath maintained at this temperature. At each 10 minutes interval juice samples in duplicate were taken out of the water bath and anthocyanin degradation as a function of heating time was monitored for 70 minutes. Effect of PPO inhibitors on plum juice anthocyanins. To study the changes in anthocyanins, plum juice was treated with 5.0% PPO extract, and different PPO inhibitors were added to give a final concentration of 0.25, 0.50 or 1.0mM for each inhibitor. Anthocyanin degradation in the presence of added PPO inhibitors was monitored for 24 hours as previously described. All the samples were maintained at 25°C in a water bath and covered to exclude room light to minimize other changes, especially ascorbic acid degradation. III. PURIFICATION OF PLUM PPO For purification of plum PPO acetone precipitate, obtained according to the method described earlier in this section, were dissolved in 0.1M sodium phosphate buffer, pH 6.0, and 0.05M calcium chloride. Calcium chloride was 37 added to precipitate pectic substances. After centrifugation at 4400 x g the supernatant, which contained crude PPO, was carefully decanted and used for subsequent purification of the enzyme. Protein content and PPO activity of the crude enzyme extract thus obtained were determined according to the methods described earlier. Ammonium sulfate fractionation Three hundred ml of crude enzyme extract, with a protein content of 0.488 mg/ml, were fractionated at 4°C with ammonium sulfate, (NH4)2SO4’ with a slight modification of the method of Park and Luh (1985). The crude extract was brought to 40% saturation by slowly adding solid (NH4)2SO4 with continuous mechanical stirring. After 30 minutes, the protein precipitate was separated by centrifugation at 8,000 x g for 15 minutes. The supernatant was carefully decanted and brought to 85% saturation by adding (NH4)2SO4 as previously described. The precipitated proteins were separated by centrifugation at 20,000 x g for 20 minutes. The precipitate was dissolved in 12 ml of cold 0.1M sodium phosphate buffer pH 6.0, and dialyzed at 4°C in the same buffer in a dialysis tubing D1616-2 (Spectra/Por 4 Dialysis Membrane Tubing, Baxter Scientific Products, Los Angles, CA). Gentle stirring was continued throughout the dialysis process. Dialysis was continued for 24 hours with three changes of the same buffer. Protein content and the activity of the dialyzed PPO was measured to determine the yield and degree of purification of PFC. Ion-exchange chromatography Ion-exchange chromatography was carried out using diethylaminoethyl (DEAE)-cellulose with a capacity of 0.9 meq/g (Sigma Corp., St. Louis, MO). The 38 DEAE-cellulose was prepared according to the method provided by the manufacturer. Twenty-five grams DEAE—cellulose were suspended in 5 volumes of deionized distilled water, stirred and allowed to settle for 45 minutes. 'Fines' were decanted twice during this time. Volume of the settled resin (column volume or CV) was measured to be used for successive washing solution volumes. The resin was first suspended in 2 CV of 0.1M NaOH containing 0.5M NaCl. The slurry was poured into a Buchner funnel (volume = 3CV) while applying gentle suction. This was followed by washing with 3 CV of deionized distilled water. The resin was then suspended in 5 CV of 0.75M HCl, poured into Buchner funnel with gentle suction being applied. Resin was first washed with 1 CV of fresh 0.75M HCl and then with 3 CV of deionized water. DEAE-cellulose prepared with the above method was carefully packed into the chromatography column (2.5 x 60 cm, Sigma Corp., St. Louis, MO) and washed with deionized water. The packed column was equilibrated at 4°C for 24 hours with the starting buffer (0.01M sodium phosphate, pH 6.0). After equilibration, 3.0 ml of enzyme sample from the (NH4)ZSO4 fractionation, containing 3.175 mg protein/ml, was loaded to the column. Column was eluted against a linear gradient of 150 ml of 0.01M sodium phosphate buffer (pH 6.0) in a mixing chamber and 150 ml of 0.5M sodium sulfate (prepared in 0.01M of sodium phosphate, pH 6.0) in the reservoir of Gradient Former (Sigma Corp., St Louis). Flow rate of the column was 45 ml/hour. Three ml fractions were collected using a Retreiver 500 fraction collecter (IASCO Corp.), and assayed for PPO activity and protein content. The fractions containing highest PPO activity were pooled and concentrated by freeze-drying for further chromatography on Sephadex G-100. 39 Gel-exclusion chromatography The concentrated enzyme from DEAE-cellulose eluents was chromatographed on Sephadex G-100 at 4°C. Sephadex G-100 was prepared by soaking in excess 0.1M sodium phosphate buffer, pH 6.0, for 72 hours with occasional stirring. Gentle stirring was done without magnetic stirrer to minimize formation of 'fines'. After 72 hours 'fines' were carefully decanted off from the top of the gel. The gel was de-aerated under low vacuum for about 15 minutes and carefully packed in a column (2.5 X 60 cm, Sigma Corp., St. Louis, M0) to a height of 50 cm. The column was equilibrated and washed with the same buffer for 48 hours. A 2.5 ml sample of the concentrated enzyme solution, having a protein content of 2.476 mg/ml, was loaded gently at the top of the gel bed and eluted with 0.1M sodium phosphate buffer pH 6.0. Three ml were collected with a flow rate of 29 ml per hour. The fractions collected were assayed for protein content and PPO activity. Fractions with highest PPO activity were pooled and concentrated by freeze-drying before subjecting them to gel-electrophoresis. Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gel-electrophoresis (PAGE) under discontinuous non- denaturing conditions was performed according to a modification of Laemmli method (Laemmli, 1979; Garfin, 1990). The gel composition was 12% polyacrylamide and 4% acrylamide for running and stacking gel, respectively: Stacking Gel: Water 6.1 ml 0.5M Tris—chloride, pH 6.8 2.5 ml Acrylamide stock solution (30%T) 1.3 ml 10% SDS ' 0.1 ml 40 Resolving Gel: Water 4.85 ml 1.5M Tris-chloride, pH 8.8 2.50 ml 10% SDS 0.10 ml Acrylamide/bis 2.50 ml 10% ammonium persulfate 50 pl TEMED 5 pl The gels were cast in a mini-gel apparatus, Mini-Protean® (Bio-Rad Labs). The electrode buffer consisted of 0.025M Tris-HCl and 0.192M glycine, pH 8.3. The PPO solution was prepared by dissolving concentrated enzyme, from Sephadex G-100 chromatography, in sample buffer (0.06M Tris-HCI and 0.025% bromophenol blue) in a ratio of 1 to 4. The samples (20 pl) were loaded into different wells. Electrophoresis was run for about one hour at 120 V in the stacking and at 200 V in the running gel. The electrophoresis run was stopped when the tracking dye (bromophenol blue) reached the bottom of the gel. Sodium dodecyl sulfate (SDS) PAGE was performed under discontinuous denaturing conditions as mentioned for PAGE except for the presence of SDS in the electrode and sample buffers. The samples were dissolved in sample buffer and boiled in a water bath for 10 minutes with 5% v/v mercaptoethanol (reducing condition). Samples without mercaptoethanol (non-reducing condiyions) were not boiled. Gels for PPO activity were stained with a modification to the method of Sayaverda-Sato and Montgomery (1986). The gel slabs were dipped and gently agitated in 30mM catechol in 0.1M sodium phosphate buffer (pH 6.0) and 0.05M p-phenylenediamine for 30 minutes or until bands showing PPO activity were visible. Then the slabs were rinsed with dionized distilled water and allowed to stand for 5 minutes with gentle agitation in SmM ascorbic acid solution to remove the excessive background color. The gels thus treated were stored in 50% 41 methanol. For protein, gel slabs were stained with 0.1% Coomassie brilliant blue R-250 (W/V) in 40% methanol-10% acetic acid solution. The gels were de-stained by washing several times with excess of 40% methanol-10% acetic acid solution. Molecular weight of plum PPO isoenzymes was estimated by comparing the corresponding protein bands of the enzyme to those of standard protein markers. Protein markers used to estimate molecular weight were carbonic anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000), phosphorylase B (97,400), B-galacto-sidase (116,000) and myosin (205,000). RESULTS AND DISCUSSION I. CHARACTERIZATION OF PPO The cultivar difference had a significant effect with respect to PPO activity (Table 2). Stanley cultivar, which is of commercial significance and widely grown in Michigan, had the highest PPO activity, followed by Pobeda and Abundance. PPO from all other plum cultivars had an activity of less than 20% as compared to that from Stanley plums. Shiro cultivar plums which are yellow in color, showed the lowest enzyme activity. Cultivar differences have been reported to be responsible for difference in PPO activity from various sources (Sciancalepore, 1985; Coseteng and Lee, 1987: Hsu et al., 1988b). Further characterization work with respect to PPO was done with Stanley cultivar plums only. The activity of enzyme in the flesh was 3.5 times greater than in the skin. PPO extracted from the skin was slightly dark colored as compared to that from the flesh. The use of polyvinyl pyrrolidone (PVP) during the final stage of enzyme extraction had little effect on the activity of PPO. Hsu et al. (1988b) also reported that PVP was not very effective in removing phenols from the crude PPO homogenate in potatoes. Substrate specificity To determine substrate specificity of plum PPO a number of mono-, di— and tri- hydroxy phenols were tested. Table 3 shows relative PPO activity with various substrates. Plum PPO acted only on o-dihydroxy or trihydroxy phenols. 42 43 Table 2. PPO activity in different plum cultivars PPO Activity/g tissue Relative Activity* Cultivar (AA420nm/min x 10'1) (%) Stanley ' 23.125 100.00 Pobeda 12.415 53.69 Abundance 7.225 30.24 Au Roadside 4.450 19.24 Wade 3.515 14.33 La Crescent 3.176 13.70 Beauty 2.690 12.80 Underwood 2.600 11.24 Pipestone 2.465 10.65 Shiro 2.445 6.22 *Relative to Stanley cultivar 44 Table 3. Substrate specificity of plum PPO with different phenolic compounds Relative Substrate Configuration Conc.(M) PPO Activity* 4—methylcatechol o-dihydroxy 0.03 152.57 Catechol o-dihydroxy 0.03 100.00 Dopamine o-dihydroxy 0.03 81. 19 Pyrogallol tri-hydroxy 0.03 75.53 Caffeic acid o-dihydroxy 0.003 60.87 DL-DOPA o-dihydroxy 0.03 16.09 Chlorogenic acid o-dihydroxy 0.003 8.97 Gallic acid tri—hydroxy 0.03 2.00 L-tyrosine monohydroxy 0.003 0.00 Hydroquinone p-dihydroxy 0.03 0.00 Phenol monohydroxy 0.03 0.00 p-cresol monohydroxy 0.03 0.00 *Relativve to catechol as 100% 45 The substrate resulting in the highest enzyme activity was 4-methylcatechol followed by catechol, dopamine and pyrogallol. Gunata et al. (1987) also demonstrated that 4-methylcatechol was oxidized much more rapidly than any other compound tested by PPO extracted from grapes and green olives. However, these results differ from those reported by Cash et al. (1976), Halim and Montgomery (1978), Piffaut and Metche (1991) where PPO from different fruits was shown to have greater affinity for catechol. On the other hand, Lee et al. (1983) observed an extremely low (6%) activity with catechol when compared to caffeic acid as substrate for DeChaunac grapes PPO. Chlorogenic acid, a naturally occuring substrate of PPO in foods, had significantly lower affinity towards PPO as compared to some other substrates. No color changes were observed with hydroquinone, L-tyrosine, phenol or p- cresol. It appears that plum PPO possesses catecholase activity only, i.e. the primary reaction catalyzed by plum PPO is the oxidation of ortho-dihydroxy phenols. PPO has been reported to possess both catecholase and cresolase activities in cherimoya epicarp (Martinez-Cayula et al., 1988) and kiwifruit (Ong et al., 1992). Plum PPO had maximum affinity toward most dihydroxy phenols, except gallic acid and chlorogenic acid, which is similar to the previous findings for PPO from different sources (Halim and Montgomery, 1978; Roudsari et al., 1981; Sachde et al., 1989; Wesche-Ebeling and Montgomery, 1990; Fujita et al., 1991). Enzyme kinetics The Km value for plum PPO was determined to be 20mM of catechol. A Km of 3.1mM for artichoke (Zawistowski et al., 1988), 3.5mM for dates (Sachde et al., 1989), 8.3mM for green olives (Ben-Shalom et al., 1977), 7.0mM for 46 eggplant (Roudsari et al., 1981), 29mM for peaches (Jen and Kahler, 1974) and 67mM for Concord grapes (Cash et al.,1976) PPO has been reported previously. The Km is a measure of the affinity of enzyme for the substrate, with smaller values representing greater affinity. The maximum reaction velocity (Vmax) was found to be 6.529 AA420mn/min x 10'1 of catechol. A Lineweaver and Burk plot of plum PPO with catechol as substrate is shown in Figure 6. Km and Vmax values observed with catechol, caffeic acid, chlorogenic acid and 4—methylcatechol are presented in Table 4. In comparison to catechol lower km and higher Vmax values were observed with 4-methylcatechol. Whereas caffeic acid and chlorogenic acid showed higher Km and lower Vmax values than those obtained with catechol. pH optima Assay of plum PPO activity in 0.1M sodium acetate buffer between pH 4.1 and 8.1 showed an optimum pH of 6.0 for maximum enzyme activity (Figure 7) with a sharp increase between pH 4.5 and 6.0. A slightly higher pH optimum (6.3) was observed when the activity was measured in 0.1M citrate-phosphate buffer. The pH curve was characterized with a rapid decrease in activity at alkaline pH values. Only one maximum was observed between the pH range studied. According to Halim and Montgomery (1978) PPO system in fruits have been shown to be most active at or near neutral pH values. These results are in close agreement with pH optimum of 5.8 reported by Cash et al. (1976) and Reyes and Luh (1960) for PPO from Concord grapes and peaches respectively. The enzyme was almost inactive at pH 4.5 (sodium acetate buffer), which is in 'contrast to pH optima of 4.5 for Airen grapes PPO (Valero et al., 1988). Above 47 20 i y = 1.5314 + 2.9086e-2x R"2 = 0.998 16 ' 12' IN (Ahzonm/min) 0 ' I ' I ' I I I ' T 0 100 200 300 400 500 1/[s] (M) Figure 6. Lineweaver-Burk plot of plum PPO with catechol. 48 Table 4. Km and Vmax values of plum PPO with different substrates Km VIIBX Substrate (mM) (Al’s420nm/min X 10'1) 4-methylcatechol 17.3 7.46 Catechol 20.0 6.53 Cafl'eic acid 26.9 3.79 Chlorogenic acid 31.6 4.08 Percent PPO Activity 49 100‘ 60" 40‘ 20‘ + 0.1M Sodium Acetate —0— 0.1M Citrate-Phosphate ’ . .. Figure 7. pH activity profile of plum PPO in sodium acetate and citrate-phosphate buffer. 50 pH 6.0 activity of the enzyme declined slowly and in contrast to some results reported earlier for PPO from other sources (Cash et al., 1976; Jen and Kahler, 1974) plum PPO showed considerable activity at pH as high as 8.0. Figure 8 shows the pH optima of plum PPO with different substrates in sodium acetate buffer, which ranged from 5.8 to 6.4. The only appreciable difference (+0.4) as compared to pH optimum with catechol was observed with 4— methylcatechol. The pH optimum with caffeic acid as substrate was determined to be 5.8. These results are in general agreement to those reported by Lourenco et al. (1990) for PPO from palmito. Effect of temperature The optimum temperature for maximum PPO activity was 20°C (Figure 9), but the enzyme retained most of its activity (80% of the maximum) over a wide range (10 to 50°C). Above 50°C the activity declined rapidly but the enzyme was not completely inactive even at 70°C. Cash et al. (1976) and Nakamura et al. (1983) reported optimum temperatures of 25 and 30°C respectively for grape PPO. For peach PPO, the optimum temperature was 37°C (Jen and Kahler, 1974). PPO from Anna apples has been reported to be thennostable between 35 and 60°C (T rejo—Gonzalez and Sato-Valdez, 1991). Blanching of lychee fruit between 56 and 100°C has been reported to inhibit browning onset, however, this treatment also bleached anthocyanins (Aikrnen, 1960). , In this study the relative PPO activity did not dip below 50% of maximum even at 10°C, which indicates that PPO activity and thus its browning and anthocyanin degradation action could not be reduced substantially by low temperature storage alone. 51 175 + 4-Methylcatechol 150- —0"— Catechol + Caffeic Acid Q 125- .2 . ‘6 < 100- C . 2 .. 75 " E 0 . 2 a: 50" 25 " 0 fl . . . . . . 3.0 4.0 5.0 6.0 7.0 8.0 pH Figure 8. pH activity profile of plum PPO with different substrates. 52 Percent PPO Activity 40' 20 f 17 ' I ' l v u 10 20 30 40 50 60 ‘70 Temperature (°C) Figure 9. Effect of temperature on plum PPO activity. 53 Storage stability of plum PPO Effect of different storage temperatures on plum PPO was studied over a 16 week period at pH 6.0 in sodium acetate buffer (Figure 10). At 20°C, which is about room temperature, the enzyme lost its complete activity after 3 weeks. At refrigeration temperature of 2°C, plum PPO lost only about 18% of its activity during first 3 weeks, however, at the end of 16 week period less than 20% of the original activity remained. The enzyme was most stable at the freezing temperature of ~20°C and even at the end of 16 weeks storage period retained about 80% of its original activity. At this temperature the enzyme can be stored for longer periods of time as the loss of enzyme activity was less than 20% even after one year (data not shown). For spinach chloroplast PPO, Sato and Hasegawa (1976) reported a loss of less than 10% in PPO activity by one year storage at -20°C. Effect of inhibitors Figures 11, 12, 13 and 14 show the effect of different inhibitors on plum PPO activity. Among ascorbic acid series compounds, ascorbic acid at all concentrations exhibited better capabilities to control PPO induced browning as compared to D-isoascorbic acid (Figure 11). This agrees with the findings of Reyes and Luh (1960), who reported ascorbic acid to be slightly better in controlling PPO activity in Fay Elberta freestone peaches as compared to D- isoascorbic acid. However, any of these two compounds could be used to control PPO action. Ascorbic acid has been shown to be effective against PPO from Concord grapes (Cash et al., 1976), d'Anjou pears (Halim and Montgomery, 1978), eggplant (Shanna and Ali, 1980) and many other sources. On the other 54 100 o 0 so a " O a? ..>. 1 ° 3 + -20°C 3 so- . —o— 2°C :1. + 22°C 9.. H 5 40- . i A. zo- ° 0 0 ' I I ' I ' I ' I r I I ' I 0 2 4 6 8 10 12 14 16 Storage Time (Weeks) Figure 10. Effect of different storage temperatures on plum PPO activity. 55 100 ‘ 4 9t 6 1 Percent PPO Inhibition A e I + Ascorbic Acid —0— D-Isoascorbic Acid Figure 11. I r T ' r , . 0.25 0.50 0.75 1.00 Inhibitor Concentration (mM) Effect of ascorbic and D-isoascorbic acid on plum PPO activity. 56 hand, ascorbic acid was reported to have no effect on strawberry PPO even at 10mM concentration (Wesche-Ebeling and Montgomery, 1990). All the benzoic acid series compounds proved to be poor inhibitors (about 30% inhibition) of plum PPO (Figure 12). There were no significant differences in their inhibitory effect at 0.50 or 1.0mM concentration. These results for benzoic acid agree with those reported for PPO inhibition from grapes (Cash et al., 1976; Gunata et al., 1987). TheSe results, however, differ from those of Gunata et al. (1987) for grape catecholase where both vanillic and syringic acid did not inhibit the enzyme even at 2.5mM concentration. Increasing concentration of all the benzoic acid series compounds from 0.25 to 1.0mM increased their inhibitory effect between 10 to 20%. Among cinnamic acid series inhibitors, p-coumaric and ferulic acid at 1.0mM concentration showed 50 and 45% decrease in PPO activity, respectively (Figure 13), while t-cinnamic acid was not as effective as p-coumaric or ferulic acid (only 29% decrease in PPO activity). However, in contrast to benzoic acid series inhibitors all cinnamic acid series inhibitors generally showed a consistent increase in their inhibitory effect on PPO when their concentration was increased from 0.05 to 1.0mM. This differs with Gunata et al. (1987) who, for grape catechol achieved 46-71% PPO inhibition with p-coumaric and cinnamic acid. However, their inhibitor concentration was significantly higher (2.5mM) than used in the current study. Sapers et al. (1989) found that sodium cinnamate, in contrast to being added alone, was more effective in controlling browning in Granny Smith apple juice when used in combination with L-ascorbyl-6-palmitate, a fat soluble analog of ascorbic acid. Gunata et al. (1987) suggested that esterification of carboxyl group of benzoic and cinnamic acid results in “a considerable loss in their inhibitory Percent PPO Inhibition 57 100 “ + Benzoic Acid '—0— Vanillic Acid 30 ' + Syringic Acid 60 ‘ . . . 0.00 0.25 0.50 0.7 5 1.00 Inhibitor Concentration (mM) Figure 12. Effect of benzoic acid series inhibitors on plum PPO activity. 58 100 ‘ , + p-Coumaric Acid —0—' Ferulic Acid 80 " + t-Cinnamic Acid 60‘ Percent PPO Inhibition o ' I ' I ' I r I 0.00 0.25 0.50 0.75 1.00 Inhibitor Concentration (mM) Figure 13. Effect of cinnamic acid series inhibitors on plum PPO activity. 59 strength. Vamos-Vigyazo (1981) also suggested that for strong PPO inhibition aromatic acid inhibitors require a free carboxylic group substituted directly on to the benzene ring. Cinnamic acid series inhibitors are not used in food processing as frequently as some other PPO inhibitors, like ascorbic acid, citric acid, cysteine, etc. However, their use in combination with some other PPO inhibition techniques may prove useful provided they are safe fOr use in food products.‘ Figure 14 shows the effectiveness of L-cysteine, sodium diethyldithiocarbamic acid, sodium metabisulfite and thiourea in inhibiting plum PPO. All these compounds exhibited a much better ability to control PPO action as compared to benzoic acid or cinnamic acid series inhibitors. At 1.0mM concentration all these compounds resulted in complete (100%) or near-complete (above 95% with thiourea) reduction in plum PPO activity. Sodium diethyldithiocarbamic acid and L-cysteine, even at 0.25mM, proved to be extremely effective inhibitors of plum PPO. The results, for L-cysteine, sodium metabisulfite and thiourea, generally agree with those reported for Concord grape PPO (Cash et al., 1976). However, Halim and Montgomery (1978) found that a 10mM concentration of these inhibitors was needed to completely inhibit the PPO in d'Anjou pears. Since PPO contains copper as a co-factor, the irreversible inactivation of this enzyme can be effected by substances, such as thiol compounds (cysteine, thiourea, 8-hydroxyquinoline, etc.), which remove copper from the active site of the enzyme (Schwimmer, 1981). Lerner et al. (1950) observed that thiol compounds exerted most of their inhibitory action by combining with the copper required. for enzymatic activity, which could be reversed by the addition of an 60 100 ' ' :2 = 80 - ‘ 2 A z 1 a —'0— Nan-diethyldithiocarbamic Acid 2 ‘0 ' + Na-metabisulfite o -+ L-Cysteine c. -—A— Thiourea 9" 4o - e’ H D c o O ‘6 c. 20 - / 0 I" ' r I I ' I I I 0.00 0.25 0.50 0.75 1.00 Inhibitor Concentration (mM) Figure 14. Effect of L-cysteine, sodium diethyldithiocarbamic acid, sodium metabisulfite and thiourea on plum PPO activity. 61 excess of cupric ions. These compounds form intermediates with quinones and thus inhibit the formation of melanins. L-cysteine seems to be the obvious replacement for sodium metabisulflte, which has been banned for use in most of the processed food products due to its health hazards. Kahn (1985) reported that proteins, peptides and amino acids can affect PPO activity in at least two ways: by reacting with the o-quinones, products of PPO action, and by chelating the essential copper at the active site of the enzyme. Ascorbic acid, although it controls the deleterious effects of PPO action, needs to be added in large quantities due to its reducing action on quinones (Schwimmer, 1981). As more quinones are formed as a result of PPO action more ascorbic acid is needed to reduce them back to their original form. In contrast to cysteine and thiourea, ascorbic acid does not inhibit PPO directly. Heat inactivation of PPO Heat inactivation of plum PPO did not follow a true linear relationship to heating time under the condition of this experiment (Figure 15). The rate of decrease in plum PPO activity at 45, 55, 65 and 75°C appeared to follow first order kinetic, particularly during initial heating (<10 minutes). This relationship was also somewhat non-linear when plotted on log scale (log PPO activity vs. heating time; data not shown). Heating of plum PPO for 4, 20 and 30 minutes at 75, 65and 55°C, respectively, rendered this enzyme completely inactive. Ong et al. (1992) reported that kiwifruit PPO was completely inactivated at 65°C when heated for 5 minutes. PPO from McFarlin cranberries was also reported to be completely inactivated in less than 3 minutes when heated at 70°C (Chan and Yang, 1971). 62 100 a + 45°C -—0— 55°C + 65°C + 7 5°C Percent Residual Activity T‘r Y I U—T U 15 20 25 30 Time (min) Figure 15. Heat inactivation of plum PPO at different temperatures. 63 At 45°C, even after heating for 30 minutes complete inactivation of plum PPO was not achieved with the enzyme still retaining about 30% of its maximum activity. Plum PPO seems to be relatively heat sensitive when compared to PPO from some other fruits. However, these results differ from those reported for Royal Ann chenies (Benjamin and Montgomery, 1973), DeChaunac grapes (Lee et al., 1983), palmito (Lourenco et al., 1990) and strawberry (Wesche-Ebeling and Montgomery, 1990) where relatively longer periods of time were required to completely inactivate the enzyme at 75°C. In case of avocado PPO, the enzyme did not lose any activity at 50°C even after heating for 20 minutes. II. EFFECT OF PPO ON ANTHOCYANINS Total phenolics and chlorogenic acid Cultivar difference had a significant effect on total phenolics and chlorogenic acid content in plums (Table 5). Total phenolics in different cultivars ranged from 262 to 922 pg/g fruit tissue (determined as chlorogenic acid equivalent). 'Beauty' had the highest level of total phenolics and 'Stanley' the lowest. The concentration of chlorogenic acid ranged from 33 to 103 pg/g, with highest concentration found in 'Beauty' and the lowest in 'Wade'. Coseteng and Lee (1987) also reported that, in case of apples, total phenolics and chlorogenic acid concentration differed from cultivar to cultivar. In our study, Stanley plums, which are of commercial importance in Michigan, had a higher chlorogenic acid to total phenolic ratio than any other cultivar. Involvement of chlorogenic acid in anthocyanin degradation has been demonstrated by Oszmianski and Lee (1991). Pifferi and Cultera (1974) reported 64 Table 5. Total phenolics and chlorogenic acid contents in different plum cultivars Total Phenolics* Chlorogenic acid Cultivar (fig/g) (fig/g) Beauty 922 103 Pipestone 736 77 La crescent 590 88 Abundance 437 38 Pobeda 353 63 Au Roadside 339 35 Underwood 300 43 Wade 299 33 Shiro 295 46 Stanley 282 75 * as chlorogenic acid 65 that among the sweet cherry phenols, chlorogenic acid and pyrocatechol were active in anthocyanin degradation. Some researchers have shown that degree of browning is to a large extent related to PPO activity only (Wuennan and Swain, 1955; Vamos-Vigyazo and Gajzago, 1976). Others (Walker, 1962; lngle and Hyde, 1968) have reported that discoloration is related to substrate content. However, Harel et al. (1966) demonstrated that both PPO activity and substrate concentration determine the degree of browning in apples. In case of plums, it was observed that degree of browning appears to be mainly related to PPO activity. Effect of heat treatment on plum juice anthocyanins Anthocyanin pigments of plum were relatively heat stable, and only 11 and 16% loss in anthocyanins was observed (Figure 16) when heated at 65°C for 30 and 70 minutes, respectively. These results differ from those reported for anthocyanins of sunflower-hull by Mok and Hettiarachchy (1991), who found that there was no change in anthocyanins when heated at 65°C. Heating of fruit products, at temperatures necessary to inactivate PPO during processing, results in the loss of characteristic color in anthocyanin rich products. In this study anthocyanins were determined qualitatively according to the method of Skalski and Sistrunk (1973). For quantitative anthocyanin determination, a pH differential method, which is more accurate, has been recommended by Frances ( 1982). Effect of PPO inhibitors on plum juice anthocyanins The loss of anthocyanin pigments in plum juice was directly related to added PPO concentration, i.e. 10% PPO as compared with 1 and 5% resulted in 66 20" Percent Anthocyanin Loss 0 U l r I U I U ‘l I I y I 1 l 0 10 20 30 40 50 60 70 Heating Time (min) Figure 16. Effect of heat treatment on plum juice anthocyanins at 65°C. 67 maximum anthocyanin loss in plum juice over 24 hours period (Figure 17). Cash et al. (1976) reported that PPO was one of the most important factors involved in the loss of color of Concord grape juice. Effect of various PPO inhibitors (at 0.25, 0.50 and 1.00 mM cone.) in controlling anthocyanin degradation was investigated over a 24 hours period (Figure 18). Sodium metabisulfite proved to be an effective inhibitor of plum PPO at concentrations of 0.25 and 0.50mM (Figure 18 a & b). Cash et al. (1976) also reported similar results for Concord grape juice. L-cysteine (at 0.50 and 1.00mM) was extremely effective in controlling anthocyanin degradation in plum juice (Figure 18 b & c), however, at 0.25mM it did not minimize anthocyanin loss (Figure 18 a). These results are similar to those reported earlier in this study for PPO inhibition in a model system. Higher concentration (1.00mM) of inhibitors, except for L-cysteine, did not improve their effectiveness with respect to anthocyanin loss in plum juice. This differs with the results reported by Embs and Markakis (1965) who concluded that higher concentrations of sulfite are more effective for inhibition of phenol oxidase. L-cysteine would have the advantage as a replacement for sulfites of being a naturally occuring amino acid that has GRAS (Generally Regarded As Safe) status for use as a dough conditioner (Dudley and Hotchkiss, 1989). Cysteine appears to act by combining with quinones rather than reducing it as ascorbic acid does (Schwimmer, 1981). Murr and Morris (1974) suggested that cysteine, in addition to binding to quinones, results in heightened levels of protease activity, which they believed, degrades phenolase, thus contributing to the prevention of discoloration. This phenomena needs to be explored further. It was also observed that cysteine delayed PPO action as compared to other inhibitors studied. This is in agreement with Dudley and Hotchkiss (1989), who reported 68 0.17 . + Control 0-1‘ —o— 1.0% PPO E + 5.0% PPO : ”'15 —c— 10.0% PPO a . ,_, 0.14- “ d 0 § 0.13- ° .e . ° 5 w a 0012- it < ' ‘\ 0.11- 0010 ' I ' I ' I r I I I ' I o 4 s 12 16 20 24 Time (hours) Figure 17 . Effect of added PPO on the rate of anthocyanin loss in plum juice at 25°C. r“. l 1 69 0'18 . —I— Control A 0 "- —0— Sodium Mctabisulfitc ( ) E ' —0— Ascorbic Acid (AA) = 0 16 —l— Citric Acid (CA) 3 ° ,. . —D— L-Cystcinc In + AA-I-CA H 0.15“ a 1 - 3 0.14- , I g . .3 o 13- ' ‘ 1. ° , ‘1' H g 0.12- < ‘ i 0.11 - + 0.10 1 r f ' I ' I ' I ' I ' ' 0 4 8 12 16 20 24 0'18 1 —I- Control _ —o— Sodium Metablsulntc (B) 0.17 —O- Ascorbic Acid (AA) E + Citric Acid (CA) 55 0.16 - —°— L43 :3 . —+— AA+CA l 0.15- i“? .3 , - ‘f j 3 0.14- = . g 0.13 - 5 u .D 0.12- 0010 ' l ' I ' I ' I ' I ‘ fl 0 4 8 12 16 20 24 0°13 F :— Control —0— Sodium Mctablsulfltc (C) E 0.17 —0— Ascorbic Acid (AA) g + Citric Acid (CA) m 0.16 —'0 L'Cmm a . " ' + AA+CA J *3 0.15- g . - e g 0.14“ . 2 ' \ I. 0.13- 2 . 4 0.12" + 0.11 ' ' 0.10 v I ' I ' l ' I ' I ' ' o 4 8 12 16 20 24 Time (hours) Figure 18. Effect of different PPO inhibitors at 0.25 mM (A), 0.50 mM (B) and 1.00 mM (C) on anthocyanin loss in plum juice. 70 that cysteine had two effects; first, an increase in lag time for PPO activity and second, increased inhibition of melanin formation. The end-products of plum juice anthocyanin degradation settled out as polymerized brown insoluble oxidation products. Neither ascorbic acid nor citric acid, alone or in combination, at the concentrations used controlled anthocyanin loss. Poei-Langston and Wrolstadt (1981) reported an accelerated loss of anthocyanin pigments in ascorbic acid containing solutions in a model system. Davidek et al. (1990) also reported that ascorbic acid exerts a negative effect on anthocyanin stability. The reaction mechanism is not fully understood but both reactants are destroyed. They hinted that it is likely that hydrogen peroxide produced in the process of ascorbic acid degradation participates in the degradation of anthocyanins. However, these results differ from those reported by Pifferi and Cultera (1974) for sweet cherry PPO. They observed that as long as ascorbic acid was present, the anthocyanins remained unaltered, and their destruction commenced, simultaneously with the appearance of chlorogenic acid oxidation products, as soon as all the ascorbic acid was consumed. Like sulfite, ascorbate helps prevent the formation of colored polymers in more than one fashion (Schwimmer, 1969). However, Varoquaux and Sanis (1979) presented evidence that ascorbic acid neither inhibits nor activates the PPO enzyme. They suggested that, in terms of preventing browning, any measure taken to prevent disappearance of ascorbic acid would also delay browning. III. PURIFICATION OF PLUM PPO The results of the purification procedures are summarized in Table 6. Through various purification procedures an increase in specific activity for plum PPO of 36.3-fold with a 21.6% yield was achieved. 71 Table 6. Purification of plum PPO Total Total Specific Purifi- Volume Activity Protein Activity cation Yield Purification Step (ml) (units) (mg) (units/mg prot.) Fold (%) Crude Extract 300.0 182,414 146.40 1,246 0.00 100.0 40—85% Ammonium Sulfate Fraction- ated and dialyzed 12.0 113,700 38.10 2,984 2.4 62.3 DEAE-cellulose 27.0 65,962 5.20 12,685 10.2 36.2 Sephadex G-100 21.0 27,615 0.61 45,270 36.3 21.6 72 A 2.4-fold purification of plum PPO was achieved through ammonium sulfate fractionation with a yield of 62.3%. A purification fold of 2 to 8 has been reported by various workers using ammonium sulfate fractionation. (Roudsari et al., 1981; Katwa et al., 1983; Wissemann and Montgomery, 1985; Zawistowski et. al., 1988; Soderal and Soderal], 1989). DEAE-cellulose chromatography Figure 19 shows the elution pattern of PPO on DEAE-cellulose column, which resulted in a single major chromatographic form of PPO while several peaks for proteins were observed. Fraction numbers 26—34, having higher PPO activity, were pooled and concentrated by freeze-drying to be used for further purification. The enzyme was purified 10.2 fold, with a 36.2% yield of total activity. Increases in specific activity as low as 2.37 fold for eggplant PPO (Roudsari et al., 1981) to as high as 218 for carrot PPO (Soderal and Soderal], 1989) have been reported for DEAE-cellulose ion-exchange chromatography. The current results for DEAE- cellulose chromatography of plum PPO agree with most of those previously reported (Nakamura et al., 1983; Wissemann and Montgomery, 1985; Zhou and Feng, 1991). Gel-exclusion chromatography on Sephadex G-100 PPO from DEAE-cellulose eluents was further purified by chromatography on Sephadex G-100 ((Figure 20). The majority of the inactive proteins were eluted first from the column. The purification fold (36.3) and yield (21.6%) data observed with plum PPO separation on Sephadex G-100 is generally similar to that reported by Sharma and Ali (1980), Galeazzi et al. (1981), Zawistowski et al. (1988) and Lourenco et al. (1990) for PPO from different sources. Column 73 cans 2.8.80 539... 0 0 0 0 8 6 4 p . . . ' 200 I 70 Protein Content 60 PPO Activity \f' I 50 - q - u u q - 0. o. o. o. 5 4 3 2 netsgfisuzs 5.23. o...— 6.0 a 1." - Fraction Number Figure 19. Ion-exchange chromatography of plum PPO on DEAE-cellulose. 74 3.0 200 _A PPO Activity '9 Protein Content III E A - " 150 'g E 2 0 " R E ' 5 e u N r: If s 4 r 100 g :2 O a C E 1 o - 3 ‘6 ° 2 3 ~50 =- G- c. , 5 K, 0.0 fl. I: r o 0 60 70 Fraction Numbers Figure 20. Gel-exclusion chromatography of plum PPO on Sephadex G-l00. 75 chromatography on Sephadex G-100 is the most commonly used final step in the purification of PPO enzyme. Polyacrylamide gel electrophoresis (PAGE) Electrophoresis under non-denaturing conditions resolved purified PPO into three distinct bands when stained for activity with 30mM catechol (Figure 21, Lane D). PAGE of crude plum PPO showed 7 bands of PPO activity (Figure 21, Lane C). Through successive purification steps involving ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose and gel exclusion chromatography on Sephadex G-100, the plum PPO was purified to such an extent that only 3 isoenzymes were present in its purified state. All the artifacts were removed during the purification process. The enzyme was homogenous, which was confirmed by a single protein band, between protein marker bands of 116,000 and 205,000, on SDS-PAGE without mercaptoethanol. Most researchers have reported 2 to 4 isoenzymes for PPO from different fruits (Wong et al., 1971; Kahn, 1977; Smith, 1985; Smith and Montgomery, 1985; Wesche-Ebeling and Montgomery, 1990). Cano et al. (1990) reported 4 isoenzymes in mature banana PPO when the gels were incubated in catechol whereas Galeazzi et al. (1981) reported 10 isoenzymes in crude PPO from banana. Gels in Lane A and B (Figure 21) were stained for proteins. The protein bands (Lane B) corresponding to the three isoenzymes had relative mobilities (Rf values) of 0.33, 0.37 and 0.55. Molecular weight of the three sub-units of purified plum PPO was estimated in the range of 45,000 to 66,000 daltons. SDS- PAGE under non-reducing conditions, showed a single protein band between the marker protein bands of 116,000 and 205,000. Partially purified PPO from several plant species has been reported to have a molecular weight in the range of 26,000 76 Rf Stained E Stained for for protein 5 PPO activity 1 .o — 1 ' . . W 205,000 - 116 000 ' — """" 0 .8 - 07,400 66,000 - o .6 - . 45,000 0 .4 " 29,000 o .2 - 0 . 0 Figure 21. Polyacrylamide gel electrophoresis of plum PPO: SDS-PAGE of standard protein markers (Lane A) and purified PPO (Lane B), stained for protein; PAGE of crude (Lane C) and purified (Lane D) PPO stained for activity with catechol. 77 to‘180,000 (Mayer and Harel, 1979). Sherman et al. (1991) reported a molecular weight in the range of 30,000 to over 200,000 for PPO extracted from different phylogenetic species. Fujita et al. (1991) and Murata et al. (1992) estimated a molecular weight of 56,000 and 46,000 by Sephadex G-100 filtration for head lettuce and apple PPO respectively. SUMMARY AND CONCLUSIONS Summary: In this study ten different plum cultivars were evaluated for PPO activity, total phenolics and chlorogenic acid concentration. After initial evaluation with respect to PPO activity subsequent characterization work was focused on Stanley plums, which are of commercial importance in Michigan. Anthocyanin study was also done on juice extracted from Stanley plums only. The optimum conditions for PPO activity (temperature and pH), enzyme kinetics (Km and Vmax). substrate specificity, storage stability, thermal inactivation and chemical inhibition of this enzyme were investigated. The optimum pH and temperature for maximum activity of this enzyme were found to be 6.0 and 20°C, respectively. The plum PPO showed activity with 0- dihydroxyphenols only with the exception of hydroquinone. 4—methylcatechol, catechol, dopamine and caffeic acid were the most readily oxidized substrates. Km and Vmax of this enzyme were determined to be 20mM and 6.529 AA420nm/min x 10'1 with catechol as substrate. Km and Vmax of this enzyme were also determined with 4—methylcatechol, caffeic acid and chlorogenic acid. The enzyme had good storage stability (about a year) at —20°C, however, it lost its activity in less than a month when stored at room temperature. Heating at 65°C (about pasteurization temperature) required 20 minutes for complete inactivation of the enzyme but at 75°C it was inactivated in about 5 minutes. L-cysteine and sodium diethyldithiocarbamic acid (at 0.25mM) ascorbic acid and D-isoascorbic acid (at 0.50mM), sodium metabisulfite and thiourea (at 1.00mM) resulted in 78 79 complete or near complete (>90%) inhibition of plum PPO. Among cinnamic acid series compounds only p-coumaric acid and ferulic acid were somewhat effective (about 60% inhibition) in controlling PPO action. All benzoic acid series compounds proved to be poor inhibitors (<30% inhibition) of plum PPO even at 1.00mM concentration. The method for juice extraction from plum was standardized at pilot plant scale using commercial pectinase enzyme. Stability of plum juice anthocyanins was also studied. Heating plum juice at 65°C (for 70 minutes) resulted in a 16% loss of anthocyanin pigments. Changes in plum juice anthocyanins were monitored in the presence of added PPO and PPO inhibitors over a 24 hour period. Sodium metabisulfite (at 0.25mM) and L-cysteine (at 0.50mM) were effective in controlling PPO induced anthocyanin degradation. Increasing concentration of PPO inhibitors did not significantly improve the degree of anthocyanin preservation. Both ascorbic and citric acid alone or in combination did not retard anthocyanin degradation. Plum PPO was purified through ammonium sulfate fractionation, dialysis, ion-exchange chromatography on DEAE-cellulose and gel-exclusion chromatography on Sephadex G-100. A 36-fold increase in specific activity with about 22% yield were achieved through these purification techniques. PAGE of crude plum PPO separated it into 7 bands when stained for activity. Most of these bands proved to be artifacts of extraction as purified PPO on PAGE separated into 3 isoenzymes only. The molecular weight of these three sub-units of the purified enzyme on SDS-PAGE was estimated to be in the range of 45,000 to 66,000 daltons by comparing against protein standards. 80 Conclusions: The conclusions that can be drawn from this study are: 1. Stanley plums had the highest PPO activity as compared to all other cultivars. Cultivar differences also had a significant effect on the concentration of total phenolics and chlorogenic acid in plums from different cultivars. 2. Plum PPO had an apparent pH optimum of 6.0 and was very sensitive to acidic pH especially below 4.5. 3. The enzyme was active over a wide range of temperature (10-60°C) with the maximum activity observed between 20 to 30°C. 4. The plum PPO exhibited catecholase activity only, i.e. it oxidized only dihydroxyphenolic substrates. This enzyme was devoid of cresolase activity as it did not act on any monohydroxyphenols. 5. The enzyme can be kept stored for about a year at -20°C with little loss in activity. In case of intact tissue, where naturally occuring phenolic substrates do not come in direct contact with the enzyme, it can remain active over longer periods of time. 6. Plum PPOcan be strongly inhibited with ascorbic acid, L-cysteine, sodium metabisulfite, sodium diethyldithiocarbamic acid and thiourea. Since the use 10. 81 of sodium metabisulfite has been banned in some food products, L-cysteine a naturally occuring amino acid can be safely used as a replacement. Plum juice anthocyanins were relatively heat stable. A loss of about 16% was observed when heated at 65°C for 70 minutes. L-cysteine and sodium metabisulfite were effective in controlling anthocyanin degradation in plum juice. Plum PPO can be successfully purified through ammonium sulfate fractionation, ion-exchange and gel-exclusion chromatography. The purified plum PPO had three isoenzymes with molecular weight in the range of 45,000-66,000 daltons. BIBLIOGRAPHY BIBLIOGRAPHY Aikrnen, E.K. 1960. 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Standard curve for proteins 1.0 0.8 '- 0.6 '- 0.4 ~ 0.2 " 0.0 y = 6.50006-2 + 7.8846e-3x R52 = 0.997 —' ‘ I ‘ I I V ' I 40 60 80 CONCENTRATION (ug) 100 120 ABSORBANCE AT 370nm Appendix 100 total phenolics 0.20 0.15‘ 0.10“ 0.05“ y = 5.7333e-3 + 2.6314e—3x m2 = 0.999 ll. Standard curve for chlorogenic acid and 0.00 I I I I I 10 20 30 40 50 60 CHLOROGENIC ACID (pg/ml) 70 "liiiiliiiiiiiiii