Purification and characterization of avocado (Persea americana) polyphenol oxidase by affinity chromatography


Alishah M. M. , 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: 10.1080/10826068.2022.2035747
  • Title of Journal : PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
  • Keywords: Affinity chromatography, Avocado (Persea americana), characterization isolation, polyphenol oxidase, INHIBITION, PROTEINS, KINETICS

Abstract

Polyphenol oxidase (PPO) enzyme was purified from avocado (Persea americana) by ammonium sulfate precipitation 0-80%, dialysis and affinity chromatography. Characterization studies were performed with catechol (0.10 M, pH: 7.2, 37 degrees C), 4-methyl catechol (0.10 M, pH: 6.0, 37 degrees C), pyrogallol (0.02 M, pH: 8.5, 5 degrees C), chlorogenic acid (0.20 M, pH: 6.8, 10 degrees C) and caffeic acid (0.20 M, pH: 8.5, 10 degrees C), respectively. V-max and K-M values were determined for catechol (15789.96 U*mL(-1)*min(-1), 10 mM), 4-methyl catechol (6768.40 U*mL(-1)*min(-1), 2 mM), pyrogallol (6802.72 U*mL(-1)*min(-1), 4 mM), chlorogenic acid (1377.97 U*mL(-1)*min(-1), 14.29 mM) and caffeic acid (2567.24 U*mL(-1)*min(-1), 5 mM). PPO was purified as 147.73-fold and 154.00-fold by Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose-6B-L-Tyrosine-p-aminobenzoic acid, respectively. 4B isolated PPO gave two bands at 35 and 50 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 kDa and 100 kDa in Native-PAGE. 6B isolated PPO gave bands as distinctively at 50 kDa and unclearly at around 35 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 and 100 kDa in Native-PAGE. The synthesis of original 6B-affinity gel and no any study found in literature on affinity purification of avocado PPO show importance of our study.