Application of three-phase partitioning to the purification and characterization of polyphenol oxidase from antioxidant rosemary (Rosmarinus officinalis L.)

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Yüzügüllü Karakuş Y. , Kahveci B., Acemi A. , Kocak G.

International Journal of Food Engineering, vol.16, 2020 (Journal Indexed in SCI) identifier identifier

  • Publication Type: Article / Article
  • Volume: 16
  • Publication Date: 2020
  • Doi Number: 10.1515/ijfe-2020-0118
  • Title of Journal : International Journal of Food Engineering
  • Keywords: enzyme, polyphenol oxidase, purification, rosemary, three-phase partitioning, ENZYME, INHIBITION, PEROXIDASE, EXTRACTION, ORIENTALIS, PROTEINS, RECOVERY


© 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.