BACKGROUND: The extraction, purification, and characterisation of peroxidase (POD) and polyphenol oxidase (PPO) were studied for Kalipatti sapota fruit. The crude enzyme extract was partially purified by ammonium sulfate precipitation followed by BioGel P100 size exclusion and Unosphere Q anion-exchange chromatography. RESULTS: Molecular weights of 20 kDa (POD) and 24 kDa (PPO) were indicated by SDS-PAGE. A single band was observed on SDS-PAGE with a fold purity of 10.38 and 7.42 for POD and PPO, respectively. Michaelis-Menten constants for POD and PPO were 22.3 and 23.0 mmol L-1 using guaiacol and catechol as substrates. Thermal inactivation kinetics was studied in the temperature range of 60-95 °C. The crude extract of POD and PPO showed D-values of 2.2-60.2 and 1.0-35.2 min; Z-values of 18.7 ± 0.4 and 16.0 ± 0.3 °C; and activation energies (Ea ) of 128.6 and 151.0 kJ mol-1 , respectively. CONCLUSION: POD and PPO showed good stability over a wide range of pH and temperature. As reflected by Z and Ea values, the fruit matrix had no significant influence towards enzyme stability. Designing of thermal process should take into consideration D- and Z-values of the enzymes along with D- and Z-values of microorganisms to obtain a product with better shelf life.
BACKGROUND: The extraction, purification, and characterisation of peroxidase (POD) and polyphenol oxidase (PPO) were studied for Kalipatti sapota fruit. The crude enzyme extract was partially purified by ammonium sulfate precipitation followed by BioGel P100 size exclusion and Unosphere Q anion-exchange chromatography. RESULTS: Molecular weights of 20 kDa (POD) and 24 kDa (PPO) were indicated by SDS-PAGE. A single band was observed on SDS-PAGE with a fold purity of 10.38 and 7.42 for POD and PPO, respectively. Michaelis-Menten constants for POD and PPO were 22.3 and 23.0 mmol L-1 using guaiacol and catechol as substrates. Thermal inactivation kinetics was studied in the temperature range of 60-95 °C. The crude extract of POD and PPO showed D-values of 2.2-60.2 and 1.0-35.2 min; Z-values of 18.7 ± 0.4 and 16.0 ± 0.3 °C; and activation energies (Ea ) of 128.6 and 151.0 kJ mol-1 , respectively. CONCLUSION: POD and PPO showed good stability over a wide range of pH and temperature. As reflected by Z and Ea values, the fruit matrix had no significant influence towards enzyme stability. Designing of thermal process should take into consideration D- and Z-values of the enzymes along with D- and Z-values of microorganisms to obtain a product with better shelf life.