This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate. © 2012 Springer-Verlag.362185192Doonan, S., Cutler, P., General strategies (2004) Protein Purification Protocols, Vol 244. Methods in Molecular Biology™, pp. 1-13. , Cutler (eds) Humana Press Totowa 10.1385/1-59259-655-x:1Narayan, A., Madhusudhan, M., Raghavarao, K., Extraction and purification of ipomoea peroxidase employing three-phase partitioning (2008) Appl Biochem Biotechnol, 151 (2), pp. 263-272. , 10.1007/s12010-008-8185-4 10.1007/s12010-008-8185-4 1:CAS:528: DC%2BD1cXhsVSitLfEZaslavsky, B.Y., Aqueous two-phase partitioning - Physical chemistry and bioanalytical applications (1995) Ber Bunsenges Phys Chem, 99 (4), pp. 221-290. , 694-694Albertsson, P.A., (1986) Partition of Cell Particles and Macromolecules, , 3 Wiley New YorkFerreira, J.F., Santana, J.C.C., Tambourgi, E.B., The effect of pH on bromelain partition from Ananas comosus by PEG4000/phosphate ATPS (2011) Braz Arch Biol Technol, 54, pp. 125-132. , 10.1590/S1516-89132011000100017 1:CAS:528:DC%2BC3MXpvFSlt7g%3DSilveira, E., Souza Jr., M.E., Santana, J.C.C., Chaves, A.C., Porto, L.F., Tambourgi, E.B., Expanded bed adsorption of bromelain (E.C. 3.4.22.33) from Ananas comosus crude extract (2009) Braz J Chem Eng, 26, pp. 149-157. , 10.1590/S0104-66322009000100014 1:CAS:528:DC%2BD1MXjvVygtbY%3DBalls, A.K., Thompson, R.R., Kies, M.W., Bromelain. Properties and commercial production (1941) Ind Eng Chem, 33 (7), pp. 950-953. , 10.1021/ie50379a028 10.1021/ie50379a028 1:CAS:528:DyaH3MXjsFOjtg%3D%3DHeinicke, R.M., Complementary enzyme actions in the clotting of milk (1953) Science, 118 (3077), pp. 753-754. , 10.1126/science.118.3077.753 10.1126/science.118.3077.753 1:CAS:528:DyaG2cXit1Witg%3D%3DMaurer, H.R., Bromelain: Biochemistry, pharmacology and medical use (2001) Cellular and Molecular Life Sciences, 58 (9), pp. 1234-1245Secor Jr., E.R., Carson IV, W.F., Cloutier, M.M., Guernsey, L.A., Schramm, C.M., Wu, C.A., Thrall, R.S., Bromelain exerts anti-inflammatory effects in an ovalbumin-induced murine model of allergic airway disease (2005) Cellular Immunology, 237 (1), pp. 68-75. , DOI 10.1016/j.cellimm.2005.10.002, PII S0008874905002376Harrach, T., Garbin, F., Munzig, E., Eckert, K., Maurer, H.R., Bromelain: An immunomodulator with anticancer activity (1994) Eur J Pharm Sci, 2 (12), p. 164. , 10.1016/0928-0987(94)90355-7Chatsworth, O.S., Cosmetic composition containing alpha hydroxyacids, salicylic acid, and enzyme mixture of bromelain and papain (1996) Biotechnol Adv, 14 (4), p. 562Fischer, G.A., Ferrari, J.F., Santana, J.C.C., Fileti, A.M.F., Tambourgi, E.B., Aqueous two-phase systems chromatography and biochemistry characterization of a enzyme from Ananas comosus (2007) Trend Chromatogr, 3, pp. 85-95. , 1:CAS:528:DC%2BD1cXhsVyqsb3ELopes, F.L.G., Severo Júnior, J.B., Souza, R., Ehrhardt, D.D., Santana, J.C.C., Tambourgi, E.B., Concentration by membrane separation processes of a medicinal product obtained from pineapple pulp (2009) Braz Arch Biol Technol, 52, pp. 457-464. , 10.1590/S1516-89132009000200024 1:CAS:528:DC%2BD1MXmt1Wlsr8%3DLarocca, M., Rossano, R., Santamaria, M., Riccio, P., Analysis of pineapple [Ananas comosus (L.) Merr.] fruit proteinases by 2-D zymography and direct identification of the major zymographic spots by mass spectrometry (2010) Food Chem, 123 (4), pp. 1334-1342. , 10.1016/j.foodchem.2010.06.016 10.1016/j.foodchem.2010.06.016 1:CAS:528:DC%2BC3cXpt1Wqsb4%3DSigma, C.C., (2012) Catalog of Biochemical and Chemical Reagents, , Sigma-Aldrich, São PauloFileti, A.M.F., Fischer, G.A., Tambourgi, E.B., Neural modeling of bromelain extraction by reversed micelles (2010) Braz Arch Biol Technol, 53, pp. 455-463. , 10.1590/S1516-89132010000200026 1:CAS:528:DC%2BC3cXot1eisb8%3DFileti, A.M.F., Fischer, G.A., Santana, J.C.C., Tambourgi, E.B., Batch and continuous extraction of bromelain enzyme by reversed micelles (2009) Braz Arch Biol Technol, 52, pp. 1225-1234. , 10.1590/S1516-89132009000500021 1:CAS:528:DC%2BC3cXisleitA%3D%3DRabelo, A.P.B., Tambourgi, E.B., Pessoa Jr., A., Bromelain partitioning in two-phase aqueous systems containing PEO-PPO-PEO block copolymers (2004) Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 807 (1), pp. 61-68. , DOI 10.1016/j.jchromb.2004.03.029, PII S1570023204002521Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal Biochem, 72 (1-2), pp. 248-254. , 10.1016/0003-2697(76)90527-3 10.1016/0003-2697(76)90527-3 1:CAS:528:DyaE28XksVehtrY%3DOliveira, L.A., Porto, A.L.F., Tambourgi, E.B., Production of xylanase and protease by Penicillium janthinellum CRC 87M-115 from different agricultural wastes (2006) Bioresource Technology, 97 (6), pp. 862-867. , DOI 10.1016/j.biortech.2005.04.017, PII S0960852405002233Englard, S., Seifter, S., Precipitation techniques (1990) Guide to Protein Purification, , M. Deutscher (eds) Academic Press San DiegoBarros Neto, B., Scarminio, I.S., Bruns, E., (2010) Como Fazer Experimentos: Aplicações Na Ciência e Na Indústria, , 4 Bookman Porto Alegre(2005) Statistica: Data Analysis Software System, , STATSOFT Version 7.1. StatSoft, TulsaGupta, R., Bradoo, S., Saxena, R.K., Aqueous two-phase systems: An attractive technology for downstream processing of biomolecules (1999) Current Science, 77 (4), pp. 520-523Feitosa, E., Catelam, K.T., Hasmann, F.A., Johansson, H.-O., Roberto, I.C., Pessoa Jr., A., Phase diagrams of a CTAB/organic solvent/buffer system applied to extraction of enzymes by reverse micelles (2008) Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 862 (1-2), pp. 58-63. , DOI 10.1016/j.jchromb.2007.10.046, PII S1570023207007659Soares, P., Coelho, D., Mazzola, P., Silveira, E., Carneiro-Da-Cunha, M.D.G., Pessoa, Jr.A., Tambourgi, E.B., Studies on bromelain precipitation by ethanol, poly(ethylene glycol) and ammonium sulphate (2011) Chem Eng Trans, 24, p. 5. , 10.3303/CET1124164Devakate, R.V., Patil, V.V., Waje, S.S., Thorat, B.N., Purification and drying of bromelain (2009) Sep Purif Technol, 64 (3), pp. 259-264. , 10.1016/j.seppur.2008.09.012 10.1016/j.seppur.2008.09.012 1:CAS:528:DC%2BD1cXhsFamt73JUmesh Hebbar, H., Sumana, B., Raghavarao, K.S.M.S., Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes (2008) Bioresource Technology, 99 (11), pp. 4896-4902. , DOI 10.1016/j.biortech.2007.09.038, PII S0960852407007845Ratanapongleka, K., Recovery of biological products in aqueous two phase systems (2010) Int J Chem Eng Appl, 1 (2), p. 7Harris, D.P., Andrews, A.T., Wright, G., Pyle, D.L., Asenjo, J.A., The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk (1998) Bioseparation, 7 (1), pp. 31-37. , 10.1023/a:1007908703773 10.1023/A:1007908703773