Biochemical and Structural Characterization of Thai Durian Polyphenol Oxidases to Determine their Catalytic Efficiency on Browning Phenomenon

        Renowned as the ‘King of Fruit’, durian is one of the most outstanding Thai economic exports that dominates both local and international markets. Its viability depends on the high-quality of the fruit production; however, durian industry generally suffers from post-harvest enzymatic browning, a process for which mainly the polyphenol oxidase (PPO) enzyme family is responsible. Though there have been some previous studies regarding durian PPO extracts, attempts to purification, identification and characterization of durian PPOs have not been biochemically reported elsewhere at a molecular level. In this proposal, RNA from Durio zibethinus will be extracted from real Thai durian samples. cDNAs encoding durian PPOs will be cloned, heterologously expressed in Escherichia coli and purified by affinity chromatography. Stability of the expressed durian enzymes will be analyzed through Circular Dichroism. The kinetic properties of the durian PPOs and their specificity toward various phenolic substrates will be determined using UV-vis spectroscopy. Moreover, crystallization of the durian PPOs will be performed for further X-ray Crystallography determination. This work will provide insights of biochemical and structural aspects of the PPOs from Thai durian and suggest the most possible phenolic compounds that could cause drastically damage to Thai durian upon browning reaction. The knowledge gained from the proposed research is hoped to generate a better strategy for intensive durian post-harvesting management that will be beneficial for local Thai gardeners and facilitate the agricultural production of Thai durian industry in the long run.