Purification of mulberry (Morus alba L.) polyphenol oxidase by affinity chromatography and investigation of its kinetic and electrophoretic properties

Abstract

Polyphenol oxidase (PPO) was isolated from mulberry (Morus alba L.) fruit using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. The purified enzyme was migrated as a single band on native and SDS-poliacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 65 kDa. Optimum PPO activity as a function of pH and temperature was determined using catechol, 4-methyl catechol and pyrogallol as substrates. The optimum pH and temperatures values of mulberry PPO for the used three substrates ranged between the pH 4.5-8.0 and 20-15 degreesC. At the optimum pH and temperature, the K-M and V-Max values of mulberry PPO towards catechol, 4-methyl catechol and pyrogallol were determined by Lineweaver-Burk method. The values V-Max/K-M showed that mulberry has the greatest reactivity towards pyrogallol among the substrates used. On the other hand mulberry PPO showed no activity toward the monophenols, p-cresol and L-tyrosine, suggesting the absence of monophenolase (cresolase) activity. Beside the classical PPO inhibitors, for the first time the inhibitory effect of some sulfonamide compounds on the mulberry PPO activities was also tested. Sulfonamide compounds exhibited considerable inhibition on mulberry PPO enzyme.