Study on the Effects of Extraction and Purification Conditions on the Activity of Peroxidase and Polyphenol Oxidase from Chestnut Kernel and on Their Substrates

Chestnut kernel is a woody grain with high nutritional value and utilized value, but it is susceptible to Browning. Previous studies have shown that phenol is the substrate of enzymes that lead to browning. In order to investigate the substrates that that leads to browning of peroxidase (POD) and polyphenol oxidase (PPO) of chestnut kernel. The extraction and purification methods and the main substrates of POD and PPO from chestnut kernel were investigated. First, the effects on enzyme activity of different kinds of buffer, temperature, time and pH value were studied; second, the salting out purification of different concentrations and the main substrates of POD and PPO from chestnut were studied. The results were shown that POD enzyme in chestnut could be obtained by extracting with sodium dihydrogen phosphate-dibasic sodium phosphate buffer (pH 6.8) at 4°C for 5 h. PPO enzyme in chestnut could be obtained by extracting with disodium hydrogen phosphate-citric acid buffer (pH 5.4) at 4°C for 5 h. The two enzymes could be purified best by 80% ammonium sulfate, and the activity of POD and PPO enzyme had been enhanced for 60% and 190% respectively. Gallic acid might be the main substrate of POD and PPO in browning of chestnut kernel.