Three phase partitioning as a large-scale separation method for purification of a wheat germ bifunctional protease/amylase inhibitor

The technique of three phase partitioning (TPP) was used to purify a wheat germ bifunctional protease/amylase inhibitor. TPP is a relatively recent technique, which uses a combination of ammonium sulphate and t-butanol to precipitate proteins from crude extracts. The precipitated protein forms interface between the lower aqueous layer and the upper organic layer. The optimization of conditions for achieving efficient purification involved studying the effects of varying temperature, ammonium sulphate concentration and the amount of t-butanol. The ratio of t-butanol to crude extract of 1:1, temperature of 20 degreesC and 30% ammonium sulphate (w/v) gave best results. The single step of TPP led to 25-fold purification with an activity recovery of 85%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of enzyme showed considerable purification and its molecular weight was found to be 21 kDa. The results are compared with those obtained with expanded bed affinity chromatography of the same system. (C) 2001 Elsevier Science Ltd. All rights reserved.