Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Abstract

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera 'Thompson Seedless'), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol L-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis-Menten constant (K m ) and maximum velocity of the reaction (V max ) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD 400 nm /min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD 478 nm /min for D, L-DOPA, respectively. The obtained similar specificity values of V max / K m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was L-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg 2? and Cu 2? increased, while Zn 2? and K ? reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25°C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25°C, but showed significant activity loss at temperatures higher than 40°C and completely inactivation at 70°C for 10 min. Thermal inactivation of PPO showed a firstorder kinetic with an activation energy (E a ) of 146.1 ± 10.8 kJ/mol at pH 6.0.

Characterisation of polyphenol oxidase and peroxidase and the role in browning of loquat fruit

“…This is qualitatively similar to the results obtained with strawberries (Dalmadi et al 2006). By using the Lineweaver-Burk plot, the Food Sci., 33, 2015 (2) (Zheng et al 2012). The data indicated that the loquat cv.…”

“…First, some inhibitors such as citric acid and oxalic acid chelate metallic ions, copper ions for PPO and ferric ions for POD (Sun et al 2012). Second, some compounds compete with the substrates and combine so that fewer amounts of the substrates are catalysed, such as l-cysteine (Zheng et al 2012). Third, reducing agents, such as ascorbic acid, reduce the block enzymatic browning by interacting with o-quinone and other substances (Gómez-López 2002).…”

Purification, partial biochemical characterization and inactivation of polyphenol oxidase from Mexican Golden Delicious apple (Malus domestica )

“…Persimmon PPO keep along the time more than 60% of its activity at 60 and 70°C (Navarro et al, ). Other reported thermal inactivation PPO temperatures are jackfruit (90°C, <5 min) (Tao et al, ), blueberry (80°C, <2 min) (Terefe, Delon, Buckow, & Versteeg, ), Thompson seedless grape (70°C, <10 min) (Zheng, Shi, & Pan, ), Bramley's seedling apple (70°C, 15 min) (Ni Eidhin et al, ), Butter lettuce (70°C, 50 min) (Gawlik‐Dziki et al, ), and litchi (60°C, 10 min) (Mizobutsi et al, ).…”

Effect of Sun, Oven and Freeze-Drying on Anthocyanins, Phenolic Compounds and Antioxidant Activity of Black Grape (Ekşikara) (Vitis vinifera L.)

“…Cell integrity and cell membranes might be damaged during drying, depending on the drying method and pretreatments (Lewicki, 1998;Ahmad et al, 2006), and therefore polyphenols in the plant undergoing drying could be oxidised by polyphenol oxidase (PPO). The optimum temperature of grape PPO differs among grape varieties and ranges from 25 o C to 45 o C (Zheng et al, 2012;Ünal & Şener, 2014). The temperature of the sun-drying method is close to optimum temperature of grape PPO.…”