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

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 °C), 4-methyl catechol (0.10 M, pH: 6.0, 37 °C), pyrogallol (0.02 M, pH: 8.5, 5 °C), chlorogenic acid (0.20 M, pH: 6.8, 10 °C) and caffeic acid (0.20 M, pH: 8.5, 10 °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.

Graphical abstract

Keywords:

• Affinity chromatography
• Avocado (Persea americana)
• characterization isolation
• polyphenol oxidase

Acknowledgements

The authors would like to acknowledge that this paper is submitted in partial fulfilment of the requirements for PhD degree at Yildiz Technical University.

Credit authorship statement

Maryam Moeini Alishah: Formal analysis, software, resources, writing- original draft. Selinnur Yıldız: Formal analysis, software, resources, writing- original draft. Çiğdem Bilen: Formal analysis, software, resources, term, conceptualization, methodology, visualization, writing (original draft, review and editing). Emine Karakuş: Formal analysis, software, resources, term, conceptualization, methodology, writing (Contact author, original draft, review and editing). All authors read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

We gratefully acknowledge for the financial support of Yildiz Technical University Science Research Projects Foundation (Project number: FBA-2020-3789) of this work.