© 2020 Walter de Gruyter GmbH, Berlin/Boston 2020Polyphenol oxidase (PPO) has been purified from the rosemary plant (Rosmarinus officinalis L.) through three-phase partitioning (TPP) and has been biochemically characterized. The optimized TPP consisted of 50% (w/v) ammonium sulfate and equal volumes of crude extract and tert-butanol prepared at pH 6.5 and room temperature. Using this system, PPO was purified 14-fold, with 230% recovery of activity from the middle phase. The partitioned enzyme had a molecular mass of 53 kDa. The highest enzyme activity was detected at 30 °C and pH 7.0 against catechol. In substrate specificity tests, the enzyme displayed activity towards catechol, 4-methylcatechol, caffeic acid, hydroquinone, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), pyrogallol, syringaldezine, and 3,4-dihydroxy-L-phenylalanine but no activity towards L-tyrosine. The enzyme was inhibited by the common PPO inhibitors; salicylhydroxamic acid (SHAM), cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and the organic solvent dimethyl sulfoxide (DMSO). Enzyme activity increased in the presence of the organic solvents acetone, ethanol, and methanol.