Extraction conditions of polyphenol oxidase from banana peel

Polyphenol oxidase (PPO) is an enzyme containing copper, presents in various fruits and vegetables. It is responsible for the browning reactions when the cells are damaged during handling. The best conditions for extraction of polyphenol oxidase from banana peel was by using an extraction buffer containing phosphate buffer (0.05 M, pH 7), 0.01 M ascorbic acid and 0.5% polyethylene glycol, with extraction ratio 1:4 (w:v) for one minute by using blender. The enzyme activity was measured spectrophotometrically at 425 nm. PPO was studied to prevent the browning of banana peel which results in the loss of their marketability. The aim of this study was to determine the optimum conditions for polyphenol oxidase extraction from banana peel.


Introduction:
Polyphenol oxidase (PPO) (EC 1.14.18.1) is a nuclear encoded enzyme that catalyzes the phenolic compounds oxidation forming corresponding quinone intermediates that polymerize to form undesirable pigment [1].Two types of the oxidative reaction are catalyzed involving molecular oxygen: the hydroxylation of monophenols to odiphenols and the oxidation of Odiphenols to O-quinones, leading to black or brown pigments formation.Polyphenol oxidase is a coppercontaining enzyme which is probably present in all plants [2].PPO is a widely distributed enzyme which is involved in biosynthesis of melanins in animals and in browning of plants.PPO plays a great role in plant tissues browning have been purified from a number of fruit tissues and their properties studied.In order to determine how to prevent the browning which results in the loss of banana peel marketability, PPO has been widely studied in many fruits and vegetables.PPO levels in a plant increase when a plant is wounded or infected [3].Multiple forms of enzyme are shown to exist from many sources.Banana is an important food sources in the developing world and one of the most important worldwide crop.It is good nutritional and health value.This enzyme is found almost in all living Open Access organisms including plants, animals and microorganisms.PPO is involved in plant defense mechanism.When a plant bruises or cut, phenolic compounds are oxidized in the presence of oxygen forming polymeric structure preventing microbial contamination [4].PPO has been also shown to have important applications such as using in valuable added products synthesis like the substituted catechol, L-DOPA for the Parkinson's disease treatment.Other catechols have found applications as starting materials or as fine chemicals for pharmaceutical drug synthesis.PPO plays an important role as an efficient reagent for cleaning polyphenolscontaining wastewater.Considering the increasing commercial applications of PPO in various fields and the development of more effective preservation conditions and methods in order to prevent the enzymatic browning, the properties of PPO from its various sources need to be studied [5].The aim of this study was to characterize PPO from banana peel in terms of best source, extraction ratio, extraction time, pH and type of extraction buffer.

Chemicals:
Catechol, polyethylene glycol (PEG), Coomassie brilliant blue, bovine serum albumin were obtained from Sigma Co. other chemicals were supplied by BDH Chemicals.

Enzyme Extraction:
Banana fruits were purchased in the yellow-green stage of ripening a local market.All experimental procedures were carried out at 4 °C.preparation of crude extract was as follows, 25 grams of banana peel were homogenized in 100 mL of phosphate buffer (0.05M, pH 7) containing ascorbic acid (0.01 M) and (0.5%) polyethylene glycol, and extraction by using blender for 1 min.Crude extract was filtered, and then the filtrated was centrifuged at 10000 rpm for 15 min at 4 °C.and suspension was used as crude enzyme [6].

Estimation of PPO Activity:
Catechol was used as substrate to determine enzyme activity.The cuvette containing the sample contained 2.9 mL of (0.01 M) substrate in phosphate buffer (0.05 M, pH 7.0) and 0.1 ml of the enzyme.3 mL of substrate solution was used as blank sample.One unit of PPO activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001/min.Calculation of polyphenol oxidase activity was used by the following equation: Where, A2 sample is the final absorbance of the sample, A1 sample is the initial absorbance of the sample; A2 blank is the final absorbance of the control, A1 blank is the initial absorbance of the control, and t is the reaction time in minutes (3 minutes) [1].

Protein concentration determination:
Protein concentration was determined according to the method of Bradford using the standard curve of bovine serum albumin [7].

PPO Source:
Different sources were used for extraction of PPO.These sources were banana pulp, banana peel, apple, Jerusalem artichoke, potato and kiwi.All sources were washed with tab water then extraction of enzyme.The enzyme activity and concentration of PPO were determination [8].
The enzyme activity and protein concentration was determination.

Extraction time:
Used different time for extraction of PPO enzyme.Banana peel was homogenized in solution extract (0.05 M phosphate buffer pH 7, 0.01 M ascorbic acid and 0.5% polyethylene glycol) at proportion 4:1 (v:w) using blender for different periods, 0.5, 1, 3, 5 and 7 minutes.The enzyme activity and protein concentration were estimate in each period.

Type of extraction buffer:
Banana peel was homogenized with different types of buffers without ascorbic acid and polyethylene glycol for PPO extraction.These buffers are sodium acetate at concentration 0.1, 0.2 and 0.05 M at pH (4.5, 5 and 5.5 for each concentration), sodium phosphate 0.1, 0.2 and 0.05 M at pH (6, 6.5), phosphate 0.1, 0.2 and 0.05 M at pH (7 and 7.5), and Tris-HCl 0.1, 0.2 and 0.05 M at pH (8, 8.5 and 9).Also used water tap for extraction of PPO enzyme from banana peel.The activity and concentration were estimate in each treatment [8].

The effect of poly ethylene glycol, ascorbic acid and polyvinylpyrrolidone on PPO enzyme extraction:
Banana peel was homogenized with potassium phosphate (0.05 M, pH 7), and one of each from the following solutions separately: -Ascorbic acid 0.01 M -Poly ethylene glycol 0.5 % -Polyvinylpyrrolidone 0.5 % -Poly ethylene glycol 0.5 % + Ascorbic acid 0.01 M -Polyvinylpyrrolidone 0.5 % + Ascorbic acid 0.01 M Then these solution were extracted at ratio 4:1 (v:w) using blender for 1 min.The enzyme activity and protein concentration were estimate in each solution.This figure shows that polyphenol oxidase from banana pulp gave highest specific activity 1127 U/mg, while banana peel, apple, jerusalem artichoke, potato and kiwi were 911.7, 157, 454, 137 and 112 U/mg, respectively.Banana peel was used as source of PPO because it's available, waste and inexpensive.Miyawaki, [9] found that the apple was best plant source of production PPO, while Kaviya,et. al.,[10] provide that banana peel was best source for PPO.

Extraction time:
Different times for PPO extraction were determined (figure 3).The best time was after one minute with specific activity 981 U/mg, while in 0.5 min., the specific activity of enzyme was decreased and reduced to 688.4 U/mg, and the reason is the low release of enzyme from banana peel.While 3, 5 and 7 minutes showed low specific activity 503.9, 456.3 and 431.4 U/mg respectively, increasing temperature for blender, causes effect on total activity for PPO enzyme.The decrease in activity at elevated temperature degrees may change the structure of the enzyme that blocks the active sites, with denaturation of enzyme [13].This result agrees with the results of Manohan and Wai [8].

Type of extraction buffer:
The specific activity PPO was estimated after extraction using different buffer, and the results were illustrated in figure (4).These results show that potassium phosphate buffer (0.05 M, pH 7) was best extraction buffer with specific activity 300 U/mg, while other buffer with different concentration and pH were given low specific activity.pH effect on enzymatic activity and stability can explained by the fact that protein structure of an enzyme molecule is influenced by the acidity or alkalinity of the solution because of the differences in ionization state of the various amino acid residues [14].The pH of enzyme environment affects the activity of the enzyme in several ways.First each enzyme has its own optimum pH, at which the maximum enzyme activity, but the enzyme is stable within certain limits under and above the optimum.Secondly, enzyme stability is influenced by environmental pH, at extremes acidity or alkalinity the enzyme may be denatured.Thirdly, the reaction mixture pH may effects on association of the substrate with the enzyme [15].Aydemir et.al.[6], provide that fresh sample of Jerusalem artichoke was homogenized in phosphate buffer containing polyethylene glycol and ascorbic acid, to give best crude enzyme.While Kaviya et.al.[9], found that best extraction solution for extraction PPO enzyme from banana peel was containing sodium phosphate buffer, Poly vinyl pyrolidone and Triton X-100.
The sources were used in extraction of PPO showed different specific activities.asshown in Figure (1), the results of the effect of the plant source type on the specific activity of enzyme.

Fig. ( 3 )
Fig. (3): The effect of extraction time for extraction of PPO from banana peel.

Fig. ( 4 )
Fig. (4): The effect the type of extraction buffer for PPO extraction from banana peel.