EXTRACTION, PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM SORGHUM (Sorghum bicolor (L) Moench)

Abstract

The study isolated, purified and characterized polyphenol oxidase (PPO) from Sorghum bicolor with the view of studying the best processing condition of this grain and the possible use of this enzyme in industrial and medicinal processing. The enzyme was and purified by ammonium sulphate precipitation, ion-exchange chromatography and size-exclusion chromatography. The physicochemical properties of the purified PPO such as optimum pH, optimum temperature, pH stability, thermal stability, substrate specificity, kinetics parameters, effect of salts and ascorbic acid were carried out. The molecular weight was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). The final purification gave a yield of 7.33% and 12.3 % and a purification fold of 2.3 and 120 for PPO from white and yellow sorghum respectively. The PPO has Vmax and Km of 2.66 U/ml and 19.72 mM for white sorghum, 1.33 U/ml and 12.92 mM for yellow sorghum. The optimum temperature of PPO from both white and yellow sorghum were found at 40 ºC and 30 ºC respectively. The optimal pH of PPO activity was found at pH 4 and pH 7, pH 4 and pH 8 for white and yellow sorghum respectively. Apparent pKa 7.4 and 8.7 were obtained for PPO from white sorghum, and pKa 5.4, 7.4 and 8.5, for yellow sorghum respectively. The PPO residual activity were above 70% at 5 hours of incubation within the neutral pH ranges for white sorghum, while those of yellow sorghum were below 40%. Zn2+, Fe2+ and ascorbic acid showed increase inhibitory effect on PPO activity from both sorghum varieties as the concentration increases while Cu2+, Na+ and K+ activates the enzyme. Over 60 % residual activity were observed between 30-50 ºC after 60 min of incubation in white and yellow sorghum. The half-life value (t1/2) of two varieties of sorghum PPOs were found between 117.46 to 17.54 minutes for white sorghum PPO and 770.0 to 25.20 minutes for yellow sorghum PPO. The PPO of white and yellow sorghum has Z-value of 36.9 °C and 20.58 °C, with the activation energy (Ea) of 51.84 kJ/mol and 92.99 kJ/mol respectively. The average value of enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) obtained at 20 minutes of incubation and temperature 50-80 °C were respectively 49.03 kJ/mol, -129.52 J/mol-1K-1, and 92.81 kJ/mol for white sorghum PPO, and 90.1 kJ/mol, -9.29 J/mol-1K-1, and 93.37 kJ/mol for yellow sorghum PPO. The PPO from white and yellow sorghum each revealed two bands by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and were estimated as 59.84 kDa and 38.02 kDa for white, 36.22 kDa and 23.01 kDa for yellow respectively. The results established the physicochemical properties of PPO from two varieties of sorghum for optimum processing condition and also proposed the amino acid in the active site of the enzyme in both sorghum varieties.