Latent polyphenol oxidase from quince fruit pulp (Cydonia oblonga): purification,activation and some properties |
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Authors: | Esteban Orenes‐Piero Francisco García‐Carmona Alvaro Snchez‐Ferrer |
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Affiliation: | Esteban Orenes‐Piñero,Francisco García‐Carmona,Alvaro Sánchez‐Ferrer |
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Abstract: | Quince fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning in Triton X‐114 and PEG 8000/phosphate with a final ammonium sulfate fractionation between 30% and 75%, to avoid the deep browning of the enzyme due to the high amount of oxidizing substances present in the quince pulp. The clean and stable enzyme was partially purified in a latent form and could be optimally activated by the presence of 0.5 g dm?3 sodium dodecyl sulfate (SDS) with an optimum pH of 5.0. In the absence of SDS, the enzyme showed maximum activity at acid pH. The apparent kinetic parameters of the latent enzyme were determined at pH 5.0, the Vm value being 15 times higher in the presence of SDS than in its absence, whereas the KM was the same in both cases, with a value of 1.2 mmol L?1. The effect of several inhibitors was studied, tropolone being the most active with a Ki value of 4.7 µmol L?1. In addition, the effect of cyclodextrins was studied, and the complexation constant (Kc) between 4‐tert‐butylcatechol and cyclodextrins was calculated using an enzymatic method. The value obtained for Kc was 15 310 mol L?1. Copyright © 2006 Society of Chemical Industry |
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Keywords: | quince polyphenol oxidase PEG‐8000 purification method diphenolase activity TBC latent enzyme SDS tropolone cyclodextrins |
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