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 te chnique, which uses a combination of ammonium sulphate and t-butanol to pre cipitate proteins from crude extracts. The precipitated protein forms inter face between the lower aqueous layer and the upper organic layer. The optim ization of conditions for achieving efficient purification involved studyin g the effects of varying temperature, ammonium sulphate concentration and t he amount of t-butanol. The ratio of t-butanol to crude extract of 1:1, tem perature 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 recover y of 85%. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis an alysis of enzyme showed considerable purification and its molecular weight was found to be 21 kDa. The results are compared with those obtained with e xpanded bed affinity chromatography of the same system. (C) 2001 Elsevier S cience Ltd. All rights reserved.