Primary Study On The Character Of Polyphenol Oxidase And The Preservation Of Lotus Sprout

Polyphenol oxidase (PPO) is widely distributed in plants, microorganisms, animals. It is a copper-containing enzyme which has complex constructure. To fresh lotus sprout as the research object, and use the modern separation techniques with lotus root extraction and purification of PPO. At the same time, the preliminary study is taking of enzymatic properties, chemical modification and structure.With the preservation of the lotus sprout was test, the results as follows:1. Extraction, purification and purity identification of PPO in lotus sprout.PPO was extracted by phosphate buffer from lotus sprout in pre-cooling 4℃, and the optimal extracting conditions were optimized,1.0%Tween80, pH6.0, extraction time were 3h, the ratio of solid to solvent were 1:3 and the crude PPO activity was 103U/ml. The crude PPO extract was purified to a high enzymicial activity of 1416.22 U/mg, which is low activity of 21.14 U/mg in crude extract, by means of separation with ammonium sulfte 30% saturation, precipitation with ammonium sulfte 80% saturation, chromatography on DEAE 52 cellulose anion exchang and on Sephadex G 100 gel. But the total enzyme activity decreased significantly, the recovery rate of protein was only 0.27%. Using SDS-PAGE gel electrophoresis purified enzyme protein molecular weight of about 35kD.2. The enzymological properties, modification of the enzyme molecule and structure characterization of PPO from Lotus sprout.The research on the activity of PPO from lotus sprout was evaluated by spectrophotometry method using catechol as substrate. The effects of temperature, pH, inhibitors and activator on PPO activity were studied and the reaction kinetic equation was established. The results showed that the optimum temperature and pH were 30℃and pH 6.0, respectively. The Km, Vmax of kinetic equation was 0.0344mol/L,172.41 U/ml, respectively. PPO would be inactivated when heated in 70℃for 60min. Ascorbic acid, citric acid, L-Cysteine, Stannous chloride all can inhibit PPO activity, The strongest inhibitor among the above was L-Cysteine, Stannous chloride, followed by citric acid, ascorbic acid,0.2g/L L-Cysteine,Stannous chloride can inhibit PPO activity effectively. The results of the effect of metal ions on the activity of PPO showed that Ca2+ and Al3+ can inhibit the activity of PPO, the inhibitory effect of Al3+ was higher than that of Ca2+. The metal ions such as Zn2+ Fe3+, Cu2+, Mg2+ and so on can accelerate the activity of PPO.The fact that the modified PPO using DTT with lower activity indicated that disulfide bonds is essential groups for the PPO activity. The fact that the modified PPO using acetic anhydride with lower activity indicated that amino bonds are essential groups for the PPO activity. The fact that the modified PPO using DTNB show that thiol bonds has nothing to do with PPO activity; The fact that the modified PPO using methanol hydrochloric acid with lower activity indicated that carboxyl bonds has closely related with the activity of PPO, as to why are some of the modified PPO activity remains to be studied further.The composition of PPO amino acid was analysised. The number of total amino acids in PPO is about 251. The content of non-polar amino acid, polar amino acids, acidic amino acids, basic amino acids is about31.69%,61.04%,20.74% and 12.66%, respectively. The structure of PPO studied by circular dichroism showed that PPO'sα-helix conformation,β-sheet conformation and Random accounted for 17.5%, 61.8% and 20.7% respectively.3. The research of preservation on lotus sproutThe results showed that different concentration of L-cysteine, ascorbic acid, citric acid, stannous chloride have some effects on color protection of lotus sprout; Orthogonal experiment showed that the optimal color retention combinations is Citric acid 200μg/g, L-cysteine 200μg/g, stannous chloride 600μg/g, ascorbic acid 200μg/g. Blanching color protection tests showed that blanching 2 minutes in boiling water is good for color retention of lotus sprout. After 30 minutes immersion in 0.2% calcium chloride solution is good for hardness of lotus sprout. Storage experiment result of lotus sprout in room temperature showed that:color retention group of color retention agents has better effect on the lotus sprout in short-term storage; color retention group of blanching has more advantage to ensure the quality of lotus sprout if the storage time is more than 8 days.