Graduate Thesis Or Dissertation
 

Purification of strawberry polyphenol oxidase and its role in anthocyanin degradation

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  • Polyphenol oxidase (PPO) from strawberries was purified, partially characterized, and used to study its role in the degradation of anthocyanin pigments. The extraction method included the use of a buffered solution containing the phenolic binders Polyclar AT and Amberlite XAD-4. Addition of Triton X-100 resulted in an increase of the extracted activity. Higher levels of PPO activity were obtained using citrate rather than acetate in the buffer. This was possibly due to the capacity of citrate to chelate calcium ions, and, therefore, to inhibit crosslinking of the pectic polymers which would trap the enzyme. Using a Phenyl Sepharose CL-4B (PS) hydrophobic column two fractions of the enzyme, Fl and F2, were separated from the pectic material and the bulk of the 280 nm absorbing material. PP0-F1 obtained from the PS column was less hydrophobic than F2, and showed a molecular weight of 111,000 by gel filtration on Sephacryl S-300. Form Fl eluted in the void volume of an anion exchange column and did not penetrate into the running gel during electrophoresis. The binding of PP0-F1 to a Concanavalin-Agarose (ConA) column demonstrated the presence of carbohydrate associated with the enzyme. PP0-F2 showed a molecular weight of 34,500 and could be resolved by anion exchange chromatography into one large peak and a smaller one at its shoulder. Fraction F2 of the enzyme resolved into two bands during electrophoresis, and did not bind to the ConA column, Both fractions of the enzyme showed multiple banding on lithium dodecyl sulfate -- polyacrylamide gradient gels. Both fractions of the enzyme obtained from the PS column showed very high activity in the presence of D-catechin. The Michaelis constants for D-catechin for PPO fractions Fl and F2 were 0.50mM and O.4lmM respectively, and the maximum velocity 82,700 and 18,800 nmoles 0₂ per min per ml respectively. D-catechin in the presence of PP0-F1 was rapidly oxidized and the resulting solution had a maximum absorption at 390 nm. When D-catechin and PP0-F1 were combined in model systems with either pure cyanin or pelargonin, an absorbance peak at 390 nm was noticed with increased absorbance in the region of the anthocyanin pigments. After 24 hr, 50% of the pelargonidin and 60% of the cyanin was destroyed and a brown precipitate was formed. PPO-Fl seemed to oxidize cyanin at a very slow rate but did not oxidize pelargonin. It was suggested that the anthocyanin pigments were destroyed by either the direct oxidation by the quinones formed from D-catechin by PPO, or that the anthocyanin pigments were co-polymerized into the brown polymeric pigment, tannin, formed from D-catechin-quinone polymerization.
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