Studies On The Characteristecs And The Inhibition By Blocking Oxygen Of PPO From Granny Smith

Granny Smith is a kind of high quality apple with high acidity, which is suitable to produce the popular juice on international market. In the process of apple juice production, the fresh fruit is easy to occur enzymatic browning, which not only greatly influences the sensory quality, the flavour and nutritional value of the product but also is an important reason for blocking the development of our fruit juice industry. This browning is usually due to the catalytic action of the enzyme polyphenoloxidase(PPO).o-Diphenols, present in the tissue of fruits and vegetables, are oxidized by polyphenoloxidase in the presence of oxygen. This oxidation reaction results in the production of o-quinones, which subsequently polymerize with other o-quinones, phenolic substances, proteins, amino acids, etc. to produce brown pigments. These pigments are sometimes referred to as melanins. In this experiment, PPO was Purified from Granny Smith ,and its common enzyme properties were studied. The activity of PPO was inhibited by reducing oxygen concentration. The study shows that blocking oxygen is the effective method to control enzymatic browning. The results are summarized as follows:(1) PPO was extracted from Granny Smith with acetone. The crude enzyme extract was fractionated with solid ammonium sulfate of 30%-90% saturation. After dialysis, the enzyme solution was purified by sephadex G-75 column chromatography respectively. The enzyme was concentrated by PEG 6000.A7.49 fold purification of PPO was obtained.(2) The result from polyacrylamide gel electrophoresis of purified enzyme indicated that only one band was found using coomassie brilliant blue R-250 stain, and their molecular weight were 31.0kD～43.0kD.(3) The largest absorbion of the product of the reaction between PPO and catechol was atλ=410nm.(4) pH optimum was 5.0 with catechol, and temperature optimum was 16℃. The enzyme had different substrate specifities for dirrerent phenolic compounds, a maxium activity was showen with catechol, followed by chlorogenic acid, pyrogallol, DL-DOPA. The activity with tyrosine was not detected. Sodium bisulfite is the best inhibitor.(5) Trichloroacetic acid is a kind of short-stopper which can stop enzymatic reaction.