| Abstract: | Black polymers were extracted from walnut husk homogenates (Juglans regia L) in a phosphate buffer (pH 8.0) after air-bubbling for 24 h. After several purification stages, ultrafiltration and gel chromatography (Sephadex and Ultrogel), three extracts (two soluble and one insoluble) were isolated. At the same time, a synthetic DL DOPA/POD/PPO copolymer was prepared. Two walnut extracts exhibited POD and PPO activity. Both DL DOPA/POD/PPO copolymers (soluble and insoluble) only presented a POD activity. The insoluble and more complex extracts (either natural or synthetic) presented the lowest oxidase activities. The soluble walnut extract exhibited low though not negligible PPO and POD activities. It was observed using electrophoresis and gel chromatography that both enzymes were present in phenolic complexes. The high level of adsorption observed on polyacrylamide gel, as well as the elementary composition of these complexes enabled a comparison with phytomelanins. PPO and POD in the soluble copolymers (from both natural and synthetic sources) were governed by Michaelis-Menten kinetics. Apparent constants were calculated using the Lineweaver-Burk method. The use of limited proteolysis and an inhibitor showed good resistance of POD activity in walnut and synthetic copolymers and exceptional resistance of PPO activity in walnut extracts (particularly insoluble extract). |
