Short communication Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor Lalit Saxena, Bharti K. Iyer, Laxmi Ananthanarayan * Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400019, India Received 13 April 2006; received in revised form 9 September 2006; accepted 20 September 2006 Abstract The technique of three-phase partitioning (TPP) was used to purify a bifunctional amylase/protease inhibitor from ragi (Eleusine coracana). This process of purification is a potential method used for separation of proteins directly from large volumes of crude suspension. It involves the addition of a salt (ammonium sulphate) to the crude extract followed by the addition of an organic solvent (t-butanol). The addition of t-butanol, in the presence of ammonium sulphate pushes the protein out of the solution to form an interfacial precipitate layer between the lower aqueous and upper organic layers. The process was carried out in two steps. The various conditions required for attaining efficient purification of the protein fractions were optimized. It was seen that 30% ammonium sulphate saturation with 1:1 ratio of crude extract to tert-butanol gave 8.9- and 8.65-fold purification with 83% and 80% yield of amylase inhibitor and trypsin inhibitor, respectively, in step I. In TPP-step II, 60% ammonium sulphate saturation and ratio of aqueous phase to t-butanol of 1:2 gave maximum 20.1- and 16-fold purification with 39.5% and 32% yield of amylase inhibitor and trypsin inhibitor, respectively. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the inhibitor protein showed substantial purification and the molecular weight of the protein was found to be 14 kDa. # 2006 Elsevier Ltd. All rights reserved. Keywords: Three-phase partitioning; Bifunctional amylase/protease inhibitor; t-Butanol; Ammonium sulphate precipitation; SDS-PAGE 1. Introduction Plant seeds are rich sources of large number of different proteinaceous inhibitors acting on a-amylases or other polysaccharide processing enzymes and proteases [1,2,3].A number of such inhibitors have been isolated and characterized from legume seeds, cereals and tubers. Proteins that inhibit a- amylases have been isolated from plants and microorganisms. These inhibitors can have natural role in the control of endogenous a-amylase activity or in defense against pathogens and pests; certain inhibitors are reported to be antinutritional factors [4]. The area of a-amylase inhibitors of plant origin has received increasing interest during the last few years. Amylase inhibitors have been isolated and purified from several sources, particularly from cereals [5]. Cereal inhibitors are mostly bifunctional in nature (i.e. they act on both amylases and proteases). These inhibitors are generally seen to have low molecular weights (10,000–50,000 Da) and have specific action against mammalian and insect amylase and trypsin enzyme. They also play a significant role in the control of endogenous a-amylase activity and in the defense against pathogen and pests [6]. Apart from this defense mechanism, these inhibitors are valuable affinity ligands for bioseparation of industrially important proteases and amylases [7]. Amylase/ trypsin inhibitors are reported to have anti-nutritional factor and therapeutic applications [8]. Traditionally, various methods are used to purify these inhibitors by conventional methods such as salting out, ion exchange chromatography, etc. Considering the above applica- tions, it is worthwhile to develop scalable and efficient purifica- tion procedure for these inhibitors. These inhibitors are generally purified by time consuming, expensive and multi step protocol. A major effort in biochemistry today is directed towards obtaining biologically important protein in an efficient fashion [9]. The present work involves purification of the proteinaceous bifunctional amylase/trypsin inhibitor from crude extract of ragi (Eleusine coracana) using a novel technique called three-phase partitioning (TPP). TPP is an up coming bioseparation technique which employs collective operations of principles involved in numerous techniques such as salting out, isoionic precipitation, cosolvent precipitation and osmolytic and kosmotropic pre- cipitation of proteins. It is easily scaleable and can be used www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 491–495 * Corresponding author. Tel.: +91 22 24145616; fax: +91 22 24145614. E-mail address: laxmi@udct.org (L. Ananthanarayan). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.09.016