Comparison of biochemical properties of membrane-bound and soluble polyphenol oxidase from Granny Smith apple (Malus × domestica Borkh.)

• Published in: Food chemistry 2019-08, Vol.289, p.657-663
• Main Authors: Han, Qian-Yun, Liu, Fang, Li, Mo, Wang, Kun-Li, Ni, Yuan-Ying
• Format: Article
• Language: English
• Subjects: Characterization; Granny Smith apple; Molecular weight; Polyphenol oxidase; Purification
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2019.02.064

•Membrane-bound polyphenol oxidase (mPPO) was the dominant form in polyphenol oxidase.•mPPO and sPPO (soluble polyphenol oxidase) exhibited diphenolase activity.•Ethylenediaminetetraacetic acid increased the activity of mPPO.•mPPO was a monomeric protein (65 kDa) while sPPO was a dimer protein (62 kDa).•The products of browning by sPPO and mPPO were brown and light yellow in color. Polyphenol oxidase from Granny Smith apples was purified and characterized in both its soluble form (sPPO) and its membrane-bound form (mPPO). Both forms were purified by temperature-induced phase partitioning, precipitation with ammonium sulfate, and ion exchange chromatography. The specific activity of mPPO was 19.17 times that of sPPO. The optimum pH and temperature for both forms were 7.0 and 35 °C when catechol was the substrate. The Michaelis constant and maximum reaction rate for sPPO were 34.1 mM and 500 U/mL/min, whereas those for mPPO were 53 mM and 10,000 U/mL/min, respectively. The enzymes exhibited diphenolase activity, and their affinity was highest for catechol (sPPO) and 4-methylcatechol (mPPO). Inhibitors of sPPO and mPPO included ascorbic acid, glutathione, and l-cysteine. However, ethylenediaminetetraacetic acid increased the activity of mPPO. Purified sPPO was dimeric with a molecular weight of 31 kDa, whereas mPPO was monomeric with an estimated molecular weight of 65 kDa.