Biochemical properties of polyphenol oxidase purified from Sarali plum (Prunus domestica)

Abstract

AbstractEnzymatic browning, catalysed by the enzyme polyphenol oxidase in fruit and vegetables, limits the efficient use of natural resources and promote food waste. Plums are a popular fruit with consumers around the world and are considered an important raw material in the food industry. Plums are very susceptible to enzymatic browning due to their high phenolic compound content and climacteric nature. The aim of this study is to purify the polyphenol oxidase enzyme from Sarali plum (Prunus domestica) and to determine its biochemical properties, kinetic parameters, pH and thermal stability and inhibition. In this study, polyphenol oxidase enzyme was purified 22.54-fold by affinity chromatography using Sepharose-4B-l-Tyr-p-amino benzoic acid affinity gel. The purity and molecular mass of the enzyme were determined by SDS-PAGE and non-denaturing PAGE (native PAGE). The molecular mass of the enzyme was estimated to be 72.44 kDa by SDS-PAGE. The enzyme was confirmed as PPO by native PAGE as a single band. Kinetic characterization studies were conducted for both catechol and 4-methyl catechol substrates. The optimal pH and temperature for both substrates were found to be 7.0 and 20 °C, respectively. The thermal stability of PPO was investigated, and it retained about 90% of its activity for 90 min at 4 °C. The determination of KM and Vmax was carried out using the Lineweaver–Burk plot. The substrate specificity (Vmax/KM) values for catechol and 4-methyl catechol were determined as 790.91 ± 37.34 and 492.06 ± 13.75 respectively. The enzyme exhibited the best activity towards catechol substrate. IC50, Ki constant and inhibition types were determined for various anti-browning agents on PPO enzyme. Ascorbic acid, l-cysteine, citric acid, salicylic acid and tartaric acid effectively inhibited PPO activity.