BCH 223 PRAC 1a PDF

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TITTLE: Polyphenol oxidase activity of bananas (Establishing a linear progress curve.) INTRODUCTION Banana is a nutritious fruit with a pleasant flavor that is widely consumed throughout the world. It is a commercially important fruit crop in the world trade. In countries such as Costa Rica and Honduras, bananas account for more than 25% of the total export (Ortiz et al., 1998). Bananas are prone to rapid browning during handling, peeling and slicing operations and even storage, if ripening is not adequately controlled. This phenomenon lowers the fruit quality and decreases its marketability (Lozano, 2007). An enzyme is a protein molecule that is a biological catalyst that speed up the rate of reaction without itself being part of the products at the end of the reaction. Like most proteins they are synthesized by the ribosomes in the cell and they have some most remarkable characteristics such as they are regulated from a state of low activity to high activity and vice-versa and also most enzymes act specifically with only one reactant called a substrate, to produce products. The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH and temperature, its activity decreases at values above and below that point due to denaturation. For enzymes, denaturation can be defined as the loss of enough structure rendering the enzyme inactive. (Banowetz,et al.,1996)

Polyphenol oxidase is the enzyme that is found in almost all living organisms including plants, animals and microorganisms. In plants, it is involved in defence mechanism. When a plant gets a bruise or cut, certain phenolic compounds are oxidized in the presence of oxygen to form a polymeric structure which prevents microbial contamination and its catalytic action is connected to undesirable browning and off,flavour generation in stored and processed foods. (Zhao,et al.,2005)

Polyphenol oxidase (-diphenol:O2 oxidoreductase) is a copper containing enzyme commonly found in plant tissues. It catalyses the oxidation of -diphenols (e.g. catechol) to -quinones (e.g. -benzophenone). Quinones so formed are highly reactive and polymerise spontaneously to give yellow or brown products. These products can be observed when plant tissue, such as potato or apple is cut and the enzyme is exposed to atmospheric oxygen. Polyphenol oxidase is of interest with

regard to the role of phenols in the resistance of plants to infection, as a rise in the level of the enzyme is often noted in infected tissue and the polymerized products from the reaction are fungiostatic. AIM: To determine the activity of the polyphenol oxidase from a banana and its absorbance.

MATERIALS AND METHOD Establishing a linear progress curve. A banana was taken and carefully peeled. The fruit was taken and mulled in 0.1M acetate buffer pH 5.6 in a chilled mortar. The amount used was dependent on the material available; 2ml of buffer was required for 1g of tissues. It was then centrifuged on a picollo for 1 minute. The supernatant was then filtered through glass wool and store in a test tube on ice. This served as the enzyme preparation. The spectrophotometer was put zero using the catechol-buffer. Mix 0.5ml of enzyme preparation to 4.5ml of catechol-buffer in a test tube, and was immediately read at 450nm every 30 seconds for at least 5 minutes.

DIS CUSSION AND RESULTS Phenol oxidases are copper proteins of wide occurrence in nature which catalyze the aerobic oxidation of certain phenolic substrates to quinones which are autoxidized to dark brown pigments generally know-n as melanins. For discussion purposes. these enzymes are assumed to be single enzymes with broad specificity, although there is some evidence for the presence of more than one phenol oxidase in certain tissues. The phenol oxidase extracted with detergent from the pulp of the ripe banana fruit is by substrate specificity a polyphenol oxidase and is similar in this respect to the enzymes of sweet potato, tobacco, tea leaf, and Solidago virgaufrea leaf. In contrast, the enzymes from most other .sources such as potato, mushroom, and several mammalian tissues are tyrosinases (or true tyrosinases) which catalyze the oxidation of both mono- and diphlenols. Yasunobu has recently reviewed the substrate specificity of a number of phenol oxidases from various sources, both plant and animal. He concluded that these enzymes catalyze the oxidation of a wide variety of substrates, but that each individual enzyme tends to catalyze the oxidation of one particular phenol (or a particular type of phenolic compound) more readily than

others. The results reported here show that banana PPO follows this general pattern, with dopamine being the most readily oxidized substrate. Results Absorbance at 450 nm Pith Fruit

Peel Time (s) 0 0.138 0.146 0.299 30 0.145 0.380 0.350 60 0.154 0.572 0.412 90 0.163 0.716 0.484 120 0.175 0.827 0.566 150 0.189 0.904 0.640 180 0.202 0.954 0.712 210 0.216 0.980 0.782 240 0.230 1.013 0.849 270 0.244 1.031 0.904 300 0.287 1.043 0.953 Table 1 the results of the absorbance obtained on the spectrophotometer after mixing a solution of the different enzymes with the catechol-buffer every 30 seconds for 5 minutes. 1.2 1 0.8 0.6 0.4 0.2 0

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Figure 1:The charts showing the absorbance obtained on the spectrophotometer after mixing a solution of the different enzymes with the catechol-buffer every 30 seconds for 5 minutes. CONCLUSION Polyphenol oxidase has been shown to occur in the pulp and peel of the banana fruit. It can be readily extracted from the pulp and rendered soluble in buffered detergent solution. These preparations were essentially colorless, and had little or no

endogenous activity. The enzyme was further purified by acetone precipitation and chromatography on DEAE-cellulose. The soluble enzyme catalyzed the oxidation of a variety of diphenolic substrates. Km values for a number of these were determine and they indicate that dopamine is most readily oxidized. Banana polyphenol oxidase in detergent extracts was relatively stable and had optimum activity at pH 7 with dopamiiine as the substrate. The mechanism dopamiiine oxidation appears to be analogous to that for the oxidation of DOPA by tyrosinase.

REFERENCES 1. BEET, A. E. 1955. Potassium permanganate in the Kjeldahl method for the determination of nitrogen in organic substances. Nature 175: 513-14. 2. BJORKMAN, 0. AND P. HOLMGREN. 1960. Polyphenols and polyphenol oxidases in leaves of Solidago virgaurea. Physiol. Plantarum 13: 58294. 3. BONNER, W. D., JR. 1957. Soluble oxidases and their functions. Ann. Rev. Plant Physiol. 8: 42752.

5. COTZIA, G. C., I. SERLIN, AND J. J. GREENOUGH. 1954. Preparation of soluble monoamine oxidase. Science 120: 144-45. 6. DAWSON, C. R. AND W. B. TARPLEY. 1951. Copper oxidases. In: The Enzymes, Vol II, Part 1, J. B. Sumner and K. Myrback, eds. Academic Press.

SURNAME INITIALS STUDENT# COURSE CODE PRACTICAL# TITLE OXIDASE BANANAS DUE DATE

:CWAYI :H.Q :201614438 :BCH 223 :01 :POLYPHENOL ACTIVITY OF :17/08/2018...