Purification and characterisation of polyphenol oxidase from red Swiss chard (Beta vulgaris subspecies cicla) leaves

We report purification and characterisation of a polyphenol oxidase from red Swiss chard (rcPPO). Our purification procedure resulted in a 39-fold enrichment in specific activity and 17% recovery of total enzyme activity. The purified rcPPO appeared as a monomeric protein of 41 kDa, with a specific conformation conserved in the Cu2+ combining region. It was optimally active at pH 7.5 and 45 °C. It had a diphenolase substrate preference towards l-DOPA, catechol and chlorogenic acid, but also exhibited weak monophenolase one toward 4-methoxyphenol and l-tyrosine. We also found that the enzyme was activated by K+, Na+, SDS and laurouyl sarcosine, but inhibited by divalent cations including Ca2+, Cu2+. Its activity was completely inhibited by ascorbic acid, cysteine, 1,4-dithiothreitol, β-mercaptoethanol, sodium diethyldithiocarbamate, sodium metabisulphite, sodium sulphite and thiourea. This first report on the purification and characterisation of red Swiss chard PPO provides a basis for understanding and use of this enzyme.