J Sci Food Agric 1991, 55, 251-260

Biochemical Studies of Cocoa Bean Polyphenol Oxidase

Pat M Lee

Department of Chemistry, Indiana University, Shah Alam Campus in Malaysia, ITM/MUCIA, Section 17, 40000 Shah Alam, Selangor, Malaysia

Kong-Hung Lee and Mohd Ismail Abdul Karim

Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia

(Received 13 February 1990; revised version received 24 September 1990; accepted 20 October 1990)

A BSTRA C T

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purfied from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K , for catechol was 1 x M . The p H optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at p H 6 8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL-alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration >40 m M was used as substrate. The enzyme activity was inhibited by ascorbic acid, L-cysteine, sodium bisulphite and thiourea.

Key words: Cocoa bean, polyphenol oxidase, Michaelis constant.

INTRODUCTION

It has been established that polyphenol oxidase (EC 1.14.18.1; PPO) in fruits and vegetables catalyses the oxidation of phenolic substrates by molecular oxygen,

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252 P M Lee, K-H Lee, M I Abdul Karim

resulting in the formation of brown pigments (Mayer and Harel 1979; Butt 1980; Vamos-Vigyazo 1981 ; Jayaraman et a1 1982). PPO is a copper-containing enzyme and is generally of broad substrate specificity and can be inhibited by a considerable number of substances (Mayer and Harel 1979; Galeazzi and Sgarbieri 1981). PPO from various types of fruits have been isolated and have been characterized (Mayer and Harel 1979) but detail investigation on cocoa beans PPO, in particular is lacking (Lee and Lee 1989a).

Flavour studies on cocoa beans have a long history. More than 300 compounds, which are believed to have contributed to the production of the unique aroma and taste of roasted cocoa beans and their finished products, have been documented. However, it is conceivable that the interactions of amino acids or polypeptides with carbohydrates in the beans may play a role in the production of flavour products and/or the precursors of flavour products. It is also likely that flavour may develop as a result of some specific enzyme reactions on intrinsic cocoa proteins. In addition to its role in the catalytic oxidation of phenolic compounds, PPO has been implicated for its role in these reactions.

In order to gain a better understanding of the possible enzymic roles in cocoa flavour formation, we initiated the studies of enzymes of cocoa beans, particularly on polyphenol oxidase. Cocoa bean polyphenol oxidase was isolated and its properties and behaviour in solution studied.

MATERIALS AND METHODS

Ripe cocoa pods were obtained from the MARDI Research Station in Teluk Intan, Perak. Polyvinylpyrrolidone (PVP), catechol, 4-methyl catechol, 4-hydroxy- phenol, p-cresol, 3-(3-4-dihydroxy pheny1)-DL-alanine, ascorbic acid, sodium bisulphite, thiourea and L-lysine were purchased from Sigma Chemicals (St Louis, USA). Sodium dihydrogen phosphate, sodium hydroxide and the Folin-Ciocalteu reagent were purchased from E. Merck (Darmstadt, FRG). Copper(I1) sulphate penta hydrate was obtained from May and Baker (Dagenham, UK).

Isolation of PPO

Cocoa bean PPO was isolated by the method of Galeazzi et al (1981). Cocoa beans were homogenised in three volumes of 0.2 M sodium phosphate buffer, pH 6.8, containing 15 g litre-' polyvinylpyrrolidone (PVP) and 5 g litre-' triton in a Waring blender with 30 s bursts for about 5 min. Homogenates were centrifuged at 22 000 x y at 4°C for 20 min followed by filtration through glass wool. To the supernate was added slowly two to three volumes of cold acetone and the solution was then stirred at 4°C for about 20 min. The precipitate was collected by centrifugation. The precipitate was re-extracted with 0.2 M sodium phosphate buffer, pH 6.8. The solution was then dialysed for 48 h against 2 litres of 50 mM phosphate buffer, pH 6.8, with four changes of buffer. The dialysate containing the enzyme was stored at 4°C and was used for the following studies.

Cocoa bean polyphenol oxidase 253

Enzyme and protein assay

The PPO activity in the enzyme extract was assayed by the method of Palmer (1963), measuring the rate of increase in the absorbance at 410 nm at 25°C with a Hitachi double beam spectrophotometer (model U-2000). The reaction mixture contained 1.0 ml of 0.05 M catechol, various amounts of enzyme and 0.2 M sodium phosphate buffer, pH 6.8, to make a total volume of 5.0 ml. The temperature was 25 f 1°C unless otherwise specified. The rate of reaction was determined from the initial linear slopes of the activity curves. One unit of PPO activity was defined as the amount of enzyme that caused 0.001 unit change in absorbance per minute at 410 nm per ml of enzyme assay solution mixture.

Specific activity was expressed as units mg-' protein. The protein solution was filtered through activated charcoal to remove phenolic and other impurities before its concentration was determined by the Lowry method (Lowry et a1 1951).

Determination of Michaelis constants of various substrates The Michaelis constants were determined under the assay conditions described above but with various concentrations of substrates ranging from 0.005405 M

and 0.10 ml of enzyme solution (0.5 mg protein ml-I). The constants (K,) were calculated from the plots of the initial rates as a function of substrate concentration according to the method of Lineweaver-Burk (Lineweaver and Burk 1934).

Polyacrylamide gel electrophoresis SDS polyacrylamide and non-reducing SDS polyacrylamide gel electrophoresis were conducted according to the method of Laemmli and Favre (1973), 12.5% polyacrylamide gel was used. The active band of PPO was detected by incubating the non-reducing gel with 1 0 m ~ catechol, pH 6.8 for 1 h followed by 5 min incubation with 1 mM ascorbic acid. The gel was then washed with water and fixed in 30% ethanol.

pH studies The PPO activity as a function of pH was determined with a solution containing 0.1 ml enzyme solution (0.5 mg protein m1-l) and 1.0 ml of 0.02 M catechol in 0.2 M sodium phosphate buffer and 4.9 ml of 0.2 M sodium phosphate buffer of pH values ranging from 4.8-8.0 under the same assay condition as described above.

Effect of temperature The thermal stability of PPO was determined by putting 1.0ml of the enzyme solution in a test tube in a water bath pre-set at the appropriate temperatures which ranged 25580°C. An aliquot (0.10 ml) of the enzyme solution was withdrawn at appropriate time intervals and rapidly chilled in an ice bath. The PPO activity was assayed with 0.02 M catechol in 0.2 M sodium phosphate buffer, pH 6.8, as described above.

Absorption spectra The absorption spectra and the absorbance of the products of the enzymically catalysed oxidation of catechol at various times were recorded with a Hitachi (Model U 2000) spectrophotometer at 25°C.

254 P M Lee, K-H Lee, M I Abdul Karim

Substrate specificity

The substrate specificity of PPO was determined with different substrates, namely catechol, 4-methyl catechol, 3-(3,4-dihydroxy phenyl)-DL-alanine, 3-hydroxy- tyramine hydrochloride, tyrosine, p-cresol and 4-hydroxy-phenol. The enzyme activity was determined at various substrate concentrations with 0.1 ml of enzyme solution (0.5 mg m1-I) in 0.20 M phosphate buffer, pH 6.8.

Inhibitor studies

The effects of inhibitors on PPO activity were determined by reacting 0.02 M catechol with a constant amount of enzyme solution in 0.2 M sodium phosphate buffer, pH 6.8 in the presence of various concentrations of inhibitors. The inhibitors studied included ascorbic acid, sodium bisulphite, L-cysteine, thiourea and EDTA. For some inhibitors used, the enzyme reaction was characterised by a lag phase. In such cases, the activities were calculated from the rate of change in absorbance immediately after the lag phase.

RESULTS AND DISCUSSION

Cocoa bean PPO was partially purified by acetone precipitation of the homogenate followed by resuspension of the acetone powder in 0.2 M phosphate buffer and dialysis of the solution. The results are summarised in Table 1. The partial puqification afforded about a seven-fold increase in enzyme activity.

Stability of enzyme

The stability of the partially purified enzyme was monitored during storage at 4°C. It was found that the enzyme stored in 0.20 M phosphate buffer was stable for several months at 4°C. It showed only a 20% decrease in activity after about 3 months and about 40% of its activity was lost after 6 months. Thus, the enzyme solution can be stored at 4°C and can be used continuously for at least 3 months.

Polyacrylamide gel electrophoresis showed 11 major protein bands and one of the protein bands was active towards catechol. This indicates that cocoa bean PPO has an apparent molecular weight of about 30 000 amu.

Absorption spectra

Figure 1 shows the change of absorbance of the enzymic catalysed oxidation of catechol at 410 nm with time. The graph of absorbance versus time was scanned

TABLE 1 Partial purification of cocoa bean PPO

Type of extract from Total Activity Spec@ Yield Purity cocoa beans cotytedon protein ( U ) activity ("/.I

(mg) ( U m 9 - l ) ~ ~ ~

Homogenate 175 7.5 7.5 x 102 100 1 Extract of acetone 70 52 5.2 x 103 40 7

Cocoa bean polyphenol oxidase 255

0 . 0 0 0 1 0 100 300 2 00

lirrek)

Fig 1. The absorbance of an enzyme solution as a function of time after the addition of 1.0 ml40 mM catechol. Absorbance at 410 nm.

0.350

0 a P

0 300 400 500 600

Wavelength (nm)

Fig 2. Absorption spectra of oxidation products of enzymically catalysed oxidation of catechol at varioustimeintervals.After(...)2min,(---)6min,(x x x )7min,(O 0 0)8min,and(--) 9 min.

immediately after the addition of catechol to the enzyme solution. It was noted that the absorbance at 410 nm increased linearly with time for the first minute, reaching maximum absorption at about 2 min. Then followed a gradual decrease in absorbance as the reaction progressed.

In order to have a better understanding of the oxidation products of this reaction, absorption spectra at various time intervals were recorded (Fig 2). The absorption peak at 410 nm is a characteristic absorption of o-quinone (catechol alone shows a maximum absorption peak at 275 nm). In addition to this absorption peak, a shoulder appeared at 295nm whose intensity was greater than that when the enzyme was free in solution. It is interesting to observe that the intensity of the absorption at 410 nm reached its maximum value after 2 min (pronounced peak in Fig 2) and thereafter its intensity decreased with a concomitant increase in the intensity at 295 nm as the oxidation progressed. This phenomenon was also observed in the oxidation of catechol with cherry PPO (Benjamin and Montgomery 1973). The decrease in the intensity at 410 nm is possibly due either to Maillard addition of the o-quinone with reactive amino moieties in the vicinity of the active site(s) of the enzyme, or to o-quinone catalysis of the Strecker degradation of the enzyme. It is probable that the decrease in intensity at 410 nm and the increase in intensity at 295 nm as the reaction progressed may correspond to the transformation of o-quinone to o-quinone-protein adduct which may absorb at 295 nm. This will be further discussed in the section on enzyme substrate specificity.

256 P M Lee, K - H Lee, M I Abdul Karim

Effect of pH

The pH optimum for the enzymatic catalysed oxidation of catechol in phosphate buffer was found to occur at pH 6.8 which coincided with the enzyme assay condition and which is also close to the pH of the cocoa bean cotyledon. At pH >8.0, the activity decreased very rapidly. The pH activity profile of PPO was consistent with the involvement of histidine residue(s) in this oxidation reaction. This also agrees with the suggestion made by Lerch (1981) that histidine residue(s) may inhibit oxidation under alkaline solution but may activate the reaction at neutral pH. It is also interesting to note that the pH optimum of PPO of some fruits also occur in the range of pH 6.0-7.5 (Reyes and Luh 1960; Luh and Phithakpoll972; Benjamin and Montgomery 1973; Halim and Montgomery 1978; Lee et a1 1983; Tengku Adnan et a1 1986). The rapid deactivation of the enzyme at pH >8.0 is presumably attributed to one or a combination of the following possibilities : conformational change of the enzyme under alkaline condition and/or the enzyme may react more rapidly with o-quinone through Maillard reaction and/or Strecker degradation and/or o-quinone itself undergoes rapid secondary reactions which are known to be catalysed by base.

Effect of temperature

The effect of heating PPO under various temperatures for different time intervals on the enzyme activity for catechol is presented in Fig 3. Cocoa bean PPO is relatively thermostable. The for enzyme activity at 80, 70 and 60°C were 5 , 20 and 25 min respectively. Niagara grape PPO (Wissemann and Lee 1981; Lee et a1 1983), peach PPO (Wong et al 1971) and Royal Ann Cherry PPO (Benjamin and Montgomery 1973) were also found to be relatively thermostable. The

60

\ 20

I

b 5 10 15 20 25 T IME(min1

Fig 3. Effect of temperature on enzyme activity in 0.2 M sodium phosphate buffer, pH 6.8, with 20 mM catechol as substrate. The enzyme solution was incubated for various time intervals at the specified temperature and rapidly cooled in an ice-bath. The remaining enzyme activity in the mixture was determined under the standard conditions. The activities measured at 25°C were taken as 100%. Activities measured at (0) 80"C, (0) 69"C, (0) 60"C, (0) 45"C, (A) 30°C were compared with the

activity measured at 25°C.

Cocoa bean polyphenol oxidase 251

optimum temperature for enzyme activity with catechol as substrate at pH 6.8 was around 45°C. At temperatures below 45"C, the enzyme was stable for at least 1-2 h. It showed no significant loss in activity within 2 h. It is interesting to note that PPO activity is dependent not only on temperature but also on the length of exposure of the enzyme to various temperatures. At 80°C the enzyme was deactivated rapidly for the first 5 min with 50% of its activity lost, followed for 20 rnin by first order kinetic deactivation. The enzyme showed only first order kinetic deactivation after 15 min at 69°C. At 60°C the enzyme exhibited a slight activation for the first 15 rnin and deactivated gradually thereafter. It is probable that during the heating process, conformational changes of the enzyme and protein-enzyme dissociation are taking place which lead to the observed deactivation of PPO. Further work is required to clarify the effect of heat on the enzyme activity. The activation energy for the catalytic oxidation of catechol, estimated from the negative slope of the Arrhenius plot at temperatures below 45"C, was found to be 56 kJ mol-'.

Substrate specifity

In Table 2, K,, V,,, and Km/Vmax data for cocoa bean PPO reacting with various monophenols and o-diphenols with various substituents at the p-positions are summarised. The enzyme was active only on o-diphenols, it was inactive towards monophenols and 4-hydroxy-phenol. No reaction was observed for up to 2 h after mixing the enzyme with monophenols (0.001 M) and 4-hydroxy-phenol (0.02 M). This indicates that cocoa bean PPO catalyses only the oxidation of o-diphenols.

Catechol, 3-(3,4-dihydroxy pheny1)-DL-alanine and 3-hydroxytyramine hydro- chloride had similar K , values while 4-methyl-catechol had a smaller K , value. The values of V,,, and Km/Vmax showed that the enzyme has the greatest reactivity towards catechol among the substrates used with 4-methyl-catechol the second most reactive substrate. However, the enzyme is also most rapidly deactivated by catechol, as has been discussed earlier (see Fig lb), and by 4-methyl catechol, especially at concentrations > 4 0 m ~ . On the other hand, it was observed that when 50 mM 3-(3,4-dihydroxy pheny1)-DL-alanine and 3-hydroxytyraminehydro-

TABLE 2 Substrate specificity of cocoa bean PPO

Substrates

Catechol 0.01 1 3846 349 636 4-Methyl catechol 1.6 x 10-3 2511 160 062 3-(3,4-dihydroxy phenyl)-m-alanine 0.0125 1666 133 280 3-hydroxytyramine hydrochloride 0.01 1 416 43 112 p-Cresol - - - Tyrosine - - - 4-h ydrox y-phenol - - -

- no activity

258 P M Lee, K-H Lee, M I Abdul Karim

chloride were used as substrates, the enzyme showed no significant deactivation after 10 min. It is possible that the substituents on the benzene ring of these two substrates undergo an intramolecular Michaelis-type reaction more rapidly than the reaction with the enzyme itself, and produce dihydroindole-quinone which absorbs at 300 nm (Palmer 1963; Lee et a1 unpublished results). However, catechol and 4-methyl catechol cannot undergo such internal Michaelis type addition reaction. Therefore they may add on to the enzyme or themselves undergo secondary reactions to form polymeric quinones absorbing at longer wavelengths. The increase in intensity of the peak at 295 nm for both catechol and 4-methyl catechol during the course of the reaction lends some support to the suggestion that this peak may arise from the o-quinoneeprotein adduct. Furthermore, the coupling of o-quinone covalently to the amino moieties in the vicinity of or at the active site(s) of the enzyme might render the active site(s) inaccessible to the substrates. This may offer a plausible explanation for the observed deactivation of the enzyme. Wood and Ingraham (1965) suggested the formation of quinone- mushroom tyrosinase adduct as the cause of enzyme deactivation.

Effect of inhibitors

The effects of inhibitors on cocoa-bean PPO activity is shown in Table 3. EDTA, even at 10 mM, slightly activated the enzyme. This suggests that metallic ions are

TABLE 3 Effect of inhibitors on cocoa beans PPO activity

Inhibitors Lug phase Inhibition (min) ("/.I

10 r n M 0 0 1 r n M 0 0

Thiourea 10 r n M 0 89

1 r n M 0 22 0.10 r n M 0 2 0.01 r n M 0 4

10 r n M > 30 ND 1 mM 1 80 0.10 mM 0 5 0.01 mM 0 0

10 r n M > 3 100 3 r n M > 30 ND 1 r n M 3 99 0.05 r n M 0 11 0.01 r n M 0 2

10 r n M > 30 ND 1 r n M 1 56 0.01 r n M 0 I

EDTA

L-Cysteine

Ascorbic acid

Sodium bisulphite

Cocoa bean polyphenol oxidase 259

not involved in the enzyme reaction. Thiourea, sodium bisulphite, L-cysteine and ascorbic acid inhibited the enzyme reaction to various extents which were directly proportional to the concentration of inhibitors. However, with the exception of thiourea, at relatively high concentration of inhibitors such as ascorbic acid, sodium bisulphite and L-cysteine, the enzyme reactions occurred only after a lag period. The duration of the lag period was directly proportional to the concentration of inhibitor used. The lag phase observed may be due to the reduction of o-quinone to catechol by the last three inhibitors. In addition, these three inhibitors (Mayer and Hare1 1979) might inhibit enzyme reaction by reacting with the enzyme molecule. Dehydroascorbic acid, the oxidation product of ascorbic acid, can react with the amino groups in close proximity to the active site(s) of the enzyme through Strecker degradation. Cysteine and sodium bisulphite, on the other hand, may react directly with sulfhydryl groups or with other amino acid residues of the enzyme. This reaction may occur concurrently with the reduction of o-quinone. Ascorbic acid appears to be the most effective inhibitor of those studied. It caused a almost complete inhibition of enzyme activity at a concentration as low as 1 mM. Thiourea appears to be the least potent inhibitor.

CONCLUSION

Cocoa bean PPO is active towards o-diphenols only. It shows a relatively high storage stability at 4°C and it has a relatively high thermal stability. The enzyme activity had a pH optimum at 6.8 and the temperature optimum was around 45"C, when catechol was used as substrate. The Michaelis constant K , for catechol was 1 x lop2 M. The enzyme activity was inhibited by ascorbic acid, L-cysteine, sodium bisulphite and thiourea. The detail mechanism of enzyme action will be further investigated.

ACKNOWLEDGEMENT

This research was supported by the Malaysian Research Science Council, grant NO 2-07-05-06.

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