Affinity precipitation of alpha-amylase inhibitor from wheat meal by metal chelate affinity binding using cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide

A method for purifying alpha-amylase inhibitor from wheat meal based on immobilized metal affinity with a thermosensitive copolymer is developed. The studies represent the thermoprecipitation properties of the copolymers of N-isopropylacrylamide (NIPAM) with iminodiacetic acid (IDA) and 1-vinylimidazole (VI), respectively. The polymer which is obtained by the copolymerization of 1-vinylimidazole and N-isopropylacrylamide, charged with Cu(II), exhibited specific interaction of the metal ions to the protein inhibitor. The precipitation was induced by salt and the recovery of the amylase inhibitor was achieved by dissolving the inhibitor-polymer complex in imidazole buffer and subsequent precipitation of the polymer. A single family of the alpha-amylase inhibitor was recovered from the polymer with 89% yield and about fourfold purification. The SDS-PAGE pattern showed significant purification of the inhibitor. The binding of the inhibitor to the Cu(II)-polymer conjugate depends upon the Cu(II) concentration in the copolymer and also upon the concentration of the protein. The recovered polymer could be reused with reasonable efficiency. Copyright 1998 John Wiley & Sons, Inc.