A Preliminary Study On The Purification And Characterization Of Bound Peroxidases In Litchi Pericarp

Fruit quality of litchi loses rapidly after harvesting due to browning, and peroxidase plays an important role in the process of postharvest browning. The change of soluble peroxidase (SPOD) activity in the process of postharvest browning has been researched.With ripe litchi (Litchi chinensis Sonn cv. Wuye) as material, the change of bound peroxidase (BPOD) activity after harvesting was firstly measured. The separation and purification system of the litchi pericarp BPOD was explored. The litchi pericarp BPOD were preliminarily separated and purified with the developed system. Finally, the first portion of BPOD enzymatic properties were characterized. The main results were as follows:The physiological indicators related with litchi browing were measured. During 4℃and 25℃ storage, the peel browning index rose with the extension of storage time. But the flesh total soluble solids (TSS) content almost no change,and they were 16% or so. During 4℃ storage, the soluble and bound POD activities rose and then fall. The changes in isozymogram bands and activities were consistent,but the new bands did not emergenced and the original bands did not disappeared. During 25 ℃ storage, the bound POD activities rose rapidly with the process of fruits browning; the isozymogram showed that activities of portions of bound POD rose significantly.The BPOD crude enzyme solutions were extracted from litchi pericarp through homogenating litchi pericarp,abandoning filtrates (the SPOD crude enzyme solutions) and repeatedly suspending residues with additional of salt solutions containing glycerol, detergent TritonX-100. The separation characteristics of BPOD crude enzyme solutions on different chromatographic media were explored, thereby establishing the separation and purification system of litchi BPOD. Four bound isoperoxidases were purified through column chromatography of Streamline Phenyl, CM-52, Phenyl Sepharose HP and Superdex 200,respectively.The enzymatic properties of first portion of BPOD were researched. The optimum pH was 6.5 and the optimum temperature was 40℃. In the presence of H2O2, for a variety of substrates it showed activities as follows:guaiacol> 4-methylcatechol> cinnamyl alcohol> chlorogenic acid> sinapyl alcohol> catechol> ascorbic acid> gallic acid> (-)-epicatechin> hydroquinone> pyrogallol. But m-dihydroxybenzene, benzylalcohol, coniferyl alcohol and 4-methoxyphenol were no response. The Km values for H2O2, guaiacol,4-methylcatechol, cinnamyl alcohol and sinapyl alcohol were 16.3mM,26.9mM,4.5mM,30.1mM,23.7 mM respectively. The activity was inhibited by DTT, ASA, L-cysteine and Cu2+. The activity was not effected by Ca2+. The activity was enhanced by Mn2+. Furthermore, The activity was not effected by 0.1 mM EDTA, but was inhibited by 1mM EDTA. The activity was enhanced by 0.01% TritonX-100, and was inhibited slightly by 0.05% TritonX-100.