Available online at www.sciencedirect.com Journal of Chromatography B, 858 (2007) 227–233 Continuous extraction of - and -amylases from Zea mays malt in a PEG4000/CaCl 2 ATPS J.P.M. Biazus a , J.C.C. Santana b,∗ , R.R. Souza c , E. Jord˜ ao d , E.B. Tambourgi d a DEQ, CCET, Federal University of Sergipe, S˜ ao Crist ´ ov˜ ao, SE, Brazil b Department of Engineering of Chemical Systems, DESQ, School of Chemical Engineering, FEQ of State University of Campinas, UNICAMP, Av. Albert Einstein 500, Postal Code 6066, 13081-970 Campinas, SP, Brazil c DEQ, CCET, Federal University of Sergipe, University City “Prof. Jos´ e Alo´ ısio de Campos”, Av. Marechal Rondon s/n, Rosa Elze, CEP 49.100-000, S˜ ao Crist ´ ov˜ ao, SE, Brazil d DESQ-FEQ, State University of Campinas, CP 6066, CEP 13081-970, Campinas, SP, Brazil Received 3 April 2007; accepted 30 August 2007 Available online 8 September 2007 Abstract In the present work, - and -amylase enzymes from Zea mays malt were recovered by continuous extraction in a PEG/CaCl 2 aqueous two-phase system (ATPS). The influences of the flux rate (R Q ), free area of vane (A free ) and vane rotation (R V ) on enzyme recovery were studied by optimization using response surface methodology (RSM). The protein content and enzyme activity were measured from time to time in the extract and refined fluxes. RSM curves showed a squared dependence of recovery index with the R Q , A free and R V . The best system for recovering the maize malt enzymes was with low vane rotation and flux rate and high free area of vane. - and -amylases were purified 130-fold in the salt-rich phase. © 2007 Elsevier B.V. All rights reserved. Keywords: Continuous extraction; - and -amylases; PEG/CaCl 2 ATPS; Zea mays malt; Maize seeds 1. Introduction Aqueous liquid–liquid two-phase systems are formed when two polymers are dissolved together above certain concentra- tions. The most characteristic feature of these systems is that both phases are aqueous (water content 85–99%), allowing par- tition of biomacromolecules and cellular particles of diverse origin under non-denaturing conditions. Several pairs of poly- mers can be used to form aqueous two-phase systems. Many polymers form two-phase liquid–liquid systems when combined with suitable salts (e.g. phosphates or sulphates) [1,2]. The use of aqueous two-phase systems in biotechnology basi- cally exploits this varying distribution of biomaterials between the phases. These systems can be buffered and are suitable for carrying out bioconversions. The phase polymers have also been shown to have a stabilizing influence on biocatalysts; the latter are, in a way, temporarily immobilized within liquid ∗ Corresponding author. Fax: +55 19 35213910. E-mail addresses: Joana biazus@yahoo.com.br (J.P.M. Biazus), jccurvelo@yahoo.com.br (J.C.C. Santana), rrsouza@ufs.br (R.R. Souza), bete@feq.unicamp.br (E. Jord˜ ao), eliastam@feq.unicamp.br (E.B. Tambourgi). droplets. The different areas in which two-phase systems have shown potential include extractive fermentations, purification of biomolecules, cells, membranes and organelles, and biological binding assays. However, most of the systems reported so far, showed an increase in purified components in the scale-up of the processes. Nevertheless, there has been a serious attempt to employ low-cost phase components, for example, in purification of proteins on a large scale [2,3]. During recent years, the well-documented phenomenon of separation of an aqueous solution of two different water-soluble polymers into individual phases has shown widespread potential in biotechnology. A number of polymers and salts have been employed for the preparation of these bi-phase systems. The most commonly used systems have been those of poly(ethylene glycol) (PEG)/NaCl, PEG/phosphate and PEG/dextran. The molecular weight of the polymers used plays an important role in determining the characteristics of the phase system [3]. The partition coefficients of hydrolytic enzymes, - and -amylases and glucoamylase, of high purity, have been deter- mined in PEG/dextran [4,5], PEG/MgSO 4 [6] and PEG/CaCl 2 [7] batch aqueous two-phase systems (ATPS). The influence of polymer, salt and initial enzyme concentration on partition 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.08.042