INTRODUCTION:

Banana is a nutritious fruit with a pleasant flavour that is widely consumed throughout the world. It is a commercially important fruit crop in the world trade. Bananas are prone to rapid browning during handling, peeling and slicing operations and even storage if ripening is not adequately controlled. Polyphenol Oxidase (PPO), monophenol dihydroxy phenylalanine: oxygen oxidoreductase (E.C.1.14.18.1) is widely distributed in the plant kingdom. It is the enzyme responsible for catalyzing the browning of polyphenol-rich fruits and vegetables^1,2.Enzymatic browning can be commonly observed in fruits such as, bananas, apples, apricots, pears and grapes, vegetables like potatoes, mushrooms and brinjals³, and also in the seafood shrimps, spiny lobsters and crabs.

During the processing of tea, coffee and chocolate, enzymatic browning is essential for the colour and taste. In these cases, the enzyme PPO plays a very important role in oxidizing the polyphenols abundantly present.

There are three types of Polyphenol Oxidases classified according to their substrate specificities and mechanism of actions. These are Tyrosinase, Catechol Oxidase and Laccase. In animals including humans, tyrosinase is mostly found and involves in the pigmentation of skin, hair and eyes for melanin synthesis⁴. In environmental technology, the presence of hazardous phenolic compounds and their derivatives in industrial wastewaters from coal conversion, petroleum refining, wood preservation, textile, paper, food and chemical industries constitutes a big problem. Recent interest has focused on the use of Peroxidases and Polyphenol Oxidases as an enzymatic approach for the removal of phenolics from industrial effluents.

PPO derived from Musa paradisica was used as a model for the study, since it is very rich in polyphenols and has a highly active PPO which leads to browning of the peel within a few minutes of peeling. Some of the natural inhibitors are non-toxic and ubiquitous in nature. It is envisaged that this study would help to identify plants and pure biologicals capable of altering enzyme activity which would be of help for various cosmetic and therapeutic applications involving PPO.

In this present study, PPO was isolated from fruit peels of Musa paradisiaca and characterized with respect to its pH and temperature optima, maximum enzyme concentration, optimum reaction time, as well as Michaelis Menton kinetics. After identifying the ideal reaction conditions, modulation of enzyme activity has been studied with aqueous extracts of some skin-friendly plants, such as, Glycyrrhiza glabra, Rubia cordifolia,Hesperethusa crenulata, oil from the seeds of Hydnocarpus laurifolia, and the pure biochemical compounds Curcumin and Psoralen^5,6.

MATERIALS AND METHODS:

EXTRACTION AND ASSAY OF POLYPHENOL OXIDASE FROM Musa paradisiacal:

The enzyme polyphenol oxidase was extracted from the fruit of banana, by using Labsonic Sonicator (B Braun) with input of 200 W and a frequency of 15 KHZ. The sample was suspended in 50 mM sodium phosphate buffer (pH 8) and sonicated for a total of 30 min with 2 minutes burst followed by 5 min rest on ice. The ground mass was centrifuged at 10,000 rpm for 15 min and the supernatant was used for further processing. All these procedures were carried out at 4-6ºC. The concentration of protein was determined by the method of Lowry et al., (1951).Enzymatic activity was measured by the rate of change in absorbance every 15 seconds, in a UV/VIS spectrophotometer (Shimadzu Corp., Tokyo, Japan) till no further change in O. D. was observed. In a 3.00 ml reaction mix, the final concentrations were 50 mM potassium phosphate, 0.17 mM catechol, 0.070 mM L-citric acid, 0.0022 mM EDTA and 50 - 100 units of catechol oxidase freshly prepared in 0.05 M sodium phosphate buffer at pH 6.5. The reaction was conducted at 25ºC.

Influence of pH on PPO activity:

Phosphate buffer of differing pH (4-10) was prepared.The reaction was performed as described above and the change in absorbance was measured at 495nm.

Influence of temperature on PPO activity:

The assay mixture containing phosphate buffer (pH 8), substrate catechol and citric acid was initially prepared and then equilibrated to the required temperature. The enzyme assay was performed at varying temperatures (28°C, 45°C, 50°C, 55°C, 60°C, 70°C and 80°C).

Influence of incubation time on PPO activity:

The assay mixture was prepared and incubated with the enzyme for varying time periods (5-30 minutes) and the absorbance after differing time periods was measured at 495 nm.

Effect of PPO concentration on its activity:

The enzyme solutions of differing volumes (50-300 µl) and concentrations were prepared and the reaction was performed for 15 minutes at 50°C. The change in absorbance was measured at 495 nm.

Effect of substrate concentration on PPO activity:

Catechol (substrate) of different concentrations (0.025 - 1.0 mM) was prepared and the reaction was performed as described in materials and methods. The Michaelis-Menten plot was drawn and then the reciprocal values of the substrate and enzyme activity were calculated using which the Lineweaver-Burk plot was drawn. From this plot, the Km and Vmax values were determined.

Effect of various plant extractsor purifiedcompoundson enzymeactivity:

The enzyme was taken in buffer and known quantity of the compound was added and the mixture was incubated for 30 minutes for reaction between the plant extract or purified compound and PPO. After noting the time, the substrate was added to start the reaction and 15 minutes later TCA was added to stop the reaction. Absorbance was measured at 495nm using a blank containing the plant extract or the purified compound. The plant extracts used were Glycyrrhiza glabra, Rubia cordifolia and Hesperethusa crenulata. The isolated or purified compounds used were Curcumin and Psoralen. Chaulmoogra oil from the seeds of Hydnocarpus laurifolia was used after suitable dilution and emulsification.

RESULTS AND DISCUSSION:

At pH 4 and 5, there was no enzyme activity. This shows that this PPO is less stable at pH values lesser than 5 where a fast denaturation of the enzyme molecules might take place. From pH 6 onwards, there was a steady increase in activity which continued till pH 10. There was a two-fold increase in the enzyme activity as the pH increased.There was a gradual increase in the activity of the enzyme when the temperature was increased from 28°C (room temperature) to 45°C. At 50°C, the enzyme showed maximum activity. Beyond 50°C, the activity of the enzyme got lowered indicating 50°C to be the optimum temperature for the enzyme’s activity. As the incubation time was increased, the enzyme activity also increased till 30 minutes.As the enzyme concentration increased, the activity of the enzyme also increased proportionally.

The enzyme activity also depends on the affinity of the enzyme towards the substrate. Km is the substrate concentration which determines the affinity of the enzyme towards the substrate. The PPO from Musa paradisiaca used in the study appears to be a very reactive enzyme with high affinity for the substrate and reacts with any level of substrate.This set of fundamental information about the enzyme activity has thus set the base for understanding the ideal experimental conditions for elucidating the effects of certain plant extracts.

Table. 1Optimum conditions for maximum enzyme activity:

Optimum conditions	Optimum values
pH	> 10
Temperature	50˚C
Km	1.1mM
Vmax	833U/mins/mg protein
Time	>30mins
Concentration of enzyme	>1.404mg

Fig.1 Effect of pH on enzyme activity

Fig.2 Effect of enzyme concentration on its activity

When assayed with 4µg plant extract per ml assay mixture, PPO activity increased by 2.7 fold with both Glycyrrhiza glabra and Rubia cordifolia. However when the concentration was increased 10 fold, the increase in activity was only 1.35 fold and 1.93 fold respectively.At low concentrations, chaulmogra oil did not influence PPO activity while at higher concentrations there was a 10 fold activation of the enzyme.

When assayed with low concentrations of Hesperethusa crenulata extract and curcumin, there was inhibition of activity by 38% and 55% respectively. At higher concentrations of 40 µg of Hesperethusa crenulataper ml of assay mixture and 800ng of curcumin per ml of assay mixture, there was activation of PPO by 82% and 50% respectively. In the present study, it was observed that the concentration of curcumin appeared to play a very critical role since trace amounts were able to inhibit the enzyme(which would mean fairer skin) while higher concentrations were enhancing the PPO activity. The addition of Psoralen to the assay mixture resulted in a proportional increase in PPO activity by 7 fold and 15 fold in the presence of 30.8µg and 61.6µg of Psoralen per ml of assay mixture. Psoralen could increase the polyphenol oxidase activity at any concentration.

Table. 2Effect of various plant extracts/purified compounds on PPO activity

Plant extract/ purified compounds

Concentration of plant extract/ purified compounds

Percentage change in enzyme activity

Glycyrrhiza glabra

40µg/ml

4µg/ml

>193

>269

Rubia cordifolia

40µg/ml

4µg/ml

>135

Chaulmogra oil

2µl/ml

4µl/ml

<7.26

>339.46

Curcumin

0.8µg/ml

8×10^-5 µg/ml

>50

<56

Hesperethusa crenulata

40µg/ml

0.4µg/ml

>82.43

<38.14

Psoralen

30.8µg/ml

61.6µg/ml

>790

>1411

CONCLUSIONS:

The activity of the enzyme PPO used in this study is activated to different extents by different compounds giving it sufficient versatility to be activated as the situation demands. Thus, plant extracts such as Glycyrrhiza glabra, Rubia cordifolia and purified compounds such as Psoralen and chaulmoogra oilare effective in enhancing enzymatic catalysis, which would make them suitable for enhancing melanin synthesis. Other extract such as those obtained from Hesperethusa crenulataand purified compound such as curcumin are moderate in enhancement of activity. The object of this study was to identify modulators from plant-derived sources, and undoubtedly, it has been successful in recognizing a variety of plants which perform the function effectively. Activation of an enzyme can be brought about by changes in the tertiary structure of the protein molecule⁸. It needs to be emphasized here that the plants are effective in increasing activity from 50 to 1411 % over control and the most suited extract could be used depending on the specific application.

In conclusion, Polyphenol Oxidase or Tyrosinase is an exceptionally versatile enzyme and more investigations are needed for a better understanding of its physiological importance and to further define its great biotechnological potential.Reports on the effect of biological compounds on PPO are scanty, as most studies have been carried out with chemical compounds. A few natural molecules discovered to modulate PPO activity include chalcones and related compounds⁹.The results obtained in the present study reveal that a search for naturally occurring modulators can lead to the discovery of a number of active compounds. Atleast 6 natural sources have been identified in this short-term study and further intense studies could help to identify more such compounds which are safe to use on the skin.

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Received on 25.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 139-142