Purification of damson plum polyphenol oxidase by affinity chromatography and investigation of metal effects on enzyme activity


Yildiz S., BİLEN Ç. , KARAKUŞ E.

PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, 2022 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Publication Date: 2022
  • Doi Number: 10.1080/10826068.2021.2023825
  • Title of Journal : PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
  • Keywords: Characterization, damson plum, inhibition, polyphenol oxidase, purifications, SUBSTRATE-SPECIFICITY, INHIBITION, FRUIT, PROTEINS, POTATO, EXTRACTION, INDUCTION

Abstract

Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0-80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 degrees C; 0.2 M/pH: 4.5/10 degrees C; 0.01 M/pH: 6.8/5 degrees C, and 0.2 M/pH: 8.5/10 degrees C, respectively. V-max and K-M values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest V-max value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu+2 activation and Fe+2 inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.