Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant Km for catechol was 1 × 10−2 M. The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3‐(3,4‐dihydroxy phenyl)‐DL‐alanine, 3‐hydroxytyramine hydrochloride and 4‐methyl catechol but showed no activity towards tyrosine, p‐cresol, and 4‐hydroxy‐phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L‐cysteine, sodium bisulphite and thiourea.

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