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Partial purification, characterisation and thermal inactivation kinetics of peroxidase and polyphenol oxidase isolated from Kalipatti sapota ( Manilkara zapota )
Author(s) -
Vishwasrao Chandrahas,
Chakraborty Snehasis,
Ananthanarayan Laxmi
Publication year - 2017
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.8215
Subject(s) - polyphenol oxidase , guaiacol , point of delivery , chemistry , peroxidase , catechol oxidase , thermal stability , kinetics , catechol , michaelis–menten kinetics , chromatography , ammonium sulfate precipitation , enzyme , enzyme kinetics , nuclear chemistry , enzyme assay , size exclusion chromatography , botany , biochemistry , organic chemistry , biology , active site , physics , quantum mechanics
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 ( E a ) 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 E a 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. © 2017 Society of Chemical Industry