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

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Yonca Yuzugullu Karakus ◽  
Busra Kahveci ◽  
Arda Acemi ◽  
Gulden Kocak
Keyword(s):  
Enzyme Activity ◽  
Polyphenol Oxidase ◽  
Cetyltrimethylammonium Bromide ◽  
Rosmarinus Officinalis ◽  
Phase Partitioning ◽  
Three Phase ◽  
Diammonium Salt ◽  
The Common ◽  
Rosmarinus Officinalis L

AbstractPolyphenol 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.

Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

2018 ◽  
Vol 14 (9-10) ◽  
Author(s):  
Neslihan Saki ◽  
Mustafa Akin ◽  
Esma H. Alici ◽  
Gulnur Arabaci
Keyword(s):  
Ammonium Sulfate ◽  
Polyphenol Oxidase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Edible Mushroom ◽  
Bottom Phase ◽  
Phase Partitioning ◽  
Three Phase ◽  
Ammonium Sulfate Saturation ◽  
Wild Edible Mushroom

AbstractPolyphenol oxidase (PPO) from the wild edible mushroom Lepiota procera is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km – 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 °C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications.

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