Purification and Characterization of Polyphenol Oxidase from the Bud of L onicera confusa

Abstract

A polyphenol oxidase (PPO), located in the bud of Lonicera confusa, was extracted, purified and characterized. Ammonium sulfate precipitation and diethyl-aminoethanol-cellulose were used to purify the PPO 20.27-fold. The molecular mass of PPO was 28.8 kDa, as determined by size exclusion chromatography combined with multiangle laser light scattering and refractive index. The optimum pH value and temperature for PPO activity in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate were 8.5 and 15C, respectively. The enzyme was stable after 60 min at 10 and 20C, and the PPO activity remained above 90%. The Km for l-DOPA was determined to be 2.26 mM. The PPO exhibited both diphenolase and triphenolase activities. Using Vmax/Km as a specificity constant, pyrocatechol was the better substrate for PPO. Moreover, ascorbic acid was a strong inhibitor that inhibited more than 78% of PPO activity at 1 mM. Practical Applications Lonicera confusa is often used in the fields of medicine and drink production. L. confusa quickly turns brown during drying and storage. Our results may facilitate the characterization of the mechanism involved in this browning process and thus provide a strategy for controlling the browning reaction as this reaction is a serious limitation in the food industry.