Purification Of POD From Longan (Dimocarpus Longan Lour.cv.Fuyan) Fruit Pericarp And Studies On Postharvest Physiology

Peroxidase (POD) and polyphenol oxidase (PPO) play the important roles of longan browning in period of postharvest storage. POD was extracted from longan (Dimocarpus longan Lour. cv. Fuyan) fruit during its maturation with extraction buffer. The crude enzyme extract was fractionated with solid ammonium sulfate between 30% and 80% saturation, then the enzyme solution was desalted by Sephadex G-25 column chromatography, separated by DEAE-Cellulose column chromatography, hydroxyapatite (HA) column chromatography and Sephacryl S-200 HR column chromatography respectively. Four separated fractions of longan PODs were purified when detected for protein with the silver stain after 5%~20% native linear gradient flatted polyacrylarnide gel electrophoresis using semi-dry technique, and corresponded to molecular weights of 290.1KD, 232.1KD, 188.5KD and 170.7KD. Four different fractions were separated when compared with polyacrylarnide gel electrophoresis. The predominant subunit of 56.4KD was showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggested that the isoenzymes of POD which molecular weights 290.1KD, 232.1KD and 170.7KD are pentamer, tetramer and trimer respectively. It is supposed that one of the isoenzymes of POD which molecular weight is 188.5KD is a kind of heteropolymer.POD and PPO showed the same relative mobilities in isoenzyme polyacrylarnide gel electrophoresis. Two active peaks of POD were separated through DEAE-Cellulose column chromatography, the former is much higher than the latter. The four fractions of purified POD are in the former peak.Properties of the purified PODs were studied. The results showed the optimal pH of POD with guaiacol is 7.0, 8.5 and 9.0 with the substrates of 4-methytlcatechol and catechol, respectively. Moreover, the PODs showed the activities of PPO. In our preliminary experiments, with the substrates of 4-methytlcatechol and catechol, the PODs appeared different PPO kinetic characteristics. The activity with 4-rnethytlcatechol was ascending from pH6.0 to pH8.0.In the thesis, the relationship between POD, PPO and the senescence and browning of longan fruit during post-harvest handling was discussed.Some physiological aspects related to the senescence and browning of longan fruit were primarily investigated as well in this thesis.