Response Surface Methodology for the Evaluation of Lysozyme Partitioning in Poly (Vinyl Pyrrolidone) and Potassium Phosphate Aqueous Two-Phase System

Document Type : Research Article


Chemical Engineering Department, Faculty of Engineering, Shomal University, Amol, I.R. IRAN


The partitioning of lysozyme and extraction yield in an aqueous two-phase system containing Poly Vinyl Pyrrolidone (PVP) K25 and potassium phosphate were investigated as a function of weight percent of salt and PVP in the feed, temperature, and pH. To investigate partitioning behavior, the central composite design was considered using a quadratic model. According to the results of the model, the partitioning of lysozyme was mainly due to the impact of the weight percent of the salt in the feed. However, the partitioning was expanded a little following the increase in the weight percent of the polymer. Based on the results, there was an opposite relationship between the temperature and decrement in the viscosity of PVP as the increment in the former led to the decrement in the latter. Finally, the modification in the third factor was done by increasing the pH level. Before experimenting, some values were hypothesized such as 93.69% for the maximum extraction yield, 21.23% for PVP K25, 13.99% for the salt concentrations, 7.10 for the pH value and 35.57 for the temperature. The findings of the study suggest that the hypothesized values for different variables are in line with the experimental results.


Main Subjects

[1] Pahlavanzadeh H., Khayati G., Ghaemi N., Vasheghani-Farahani E., Liquid-Liquid Extraction of 2, 3-Butanediol from Fermentation Broth, Iran. J. Chem. Chem. Eng. (IJCCE), 31(2): 59-63. (2012).
[5] Asenjo J.A., Andrews B.A., Aqueous Two-Phase Systems for Protein Separation: Phase Separation and Applications, J. Chromatogr. A 1238: 1-10 (2012).
[7] Rodrigues G.D., de Lemos L.R., da Silva L.H.M., da Silva M.C.H., Application of Hydrophobic Extractant in Aqueous Two-Phase Systems for Selective Extraction of Cobalt, Nickel and Cadmium, J. Chromatogr. A, 1279: 13-19(2013).
[8] Tomasula P., Parris N., Boswell R., Moten R., Preraration of Enrichedfractions of α‐Lactalbumin and β‐Lactoglobulin from Cheese Whey Using Carbon Dioxide, J. Food Process. Preserv 22(6): 463-476 (1998).
[11] da Costa A.R., dos Reis Coimbra J.S., Ferreira L.A., Marcos J.C., Santos I.J.B., Saldaña M.D., Teixeira J.A.C., Partitioning of Bovine Lactoferrin in Aqueous Two-Phase System Containing Poly (ethylene glycol) and Sodium Citrate, Food Bioprod Process, 95: 118-124 (2015).
[13] Gavasane M.R., Gaikar V.G., Aqueous Two-Phase Affinity Partitioning of Penicillin Acylase from E. Coli in Presence of PEG-Derivatives, Enzyme Microb. Technol., 32(6): 665-675 (2003).
[14] Fox P., Kelly A., Indigenous Enzymes in Milk: Overview and Historical Aspects—Part 2, Int Dairy J.,16(6): 517-532 (2006).
[15] Hughey V., Johnson E., Antimicrobial Activity of Lysozyme Against Bacteria Involved in Food Spoilage and Food-Borne Disease, Appl. Environ. Microbiol., 53(9): 2165-2170 (1987).
[18] Alderton G., Fevold H., Direct Crystallization of Lysozyme from Egg White and Some Crystalline Salts of Lysozyme, J. Biol. Chem. 164(1): 1-5 (1946).
[19] Chiang C.-M., Ge H., Wang Z., Hoffmann A., Roeder R., Unique TATA-Binding Protein-Containing Complexes and Cofactors Involved in Transcription by RNA Polymerases II and III, The EMBO Journal, 12(7): 2749 (1993).
[20] Ghosh R., Silva S.S., Cui Z., Lysozyme Separation by Hollow-Fibre Ultrafiltration, Biochem. Eng. J., 6(1): 19-24 (2000).
[21] Guérin-Dubiard C., Pasco M., Hietanen A., del Bosque A.Q., Nau F., Croguennec T., Hen Egg White Fractionation by Ion-Exchange Chromatography, J. Chromatogr. A 1090(1): 58-67 (2005).
[22] Lee M.-H., Process for Preparing Lysozyme, Google Patents, (2006).
[23] Silvério S.C., Ferreira L.A., Martins J., Marcos J.C., Macedo E., Teixeira J., Lysozyme and Bovine Serum Albumin Partitioning in Polyethylene Glycol–Phenylalanine Conjugate Polymer/Salt Aqueous Two-Phase Systems, Fluid Phase Equilib., 322: 19-25 (2012).
[24] de Sousa R.d.C.S., dos Reis Coimbra J.S., da Silva L.H.M., da Silva M.d.C.H., Rojas E.E.G., Vicente A.A.A., thermodynamic Studies of Partitioning Behavior of Lysozyme and Conalbumin in Aqueous Two-Phase Systems, J. Chromatogr. B, 877(24): 2579-2584 (2009).
[25] Salamatinia B., Hashemizadeh I., Ahmad Zuhairi A., Alkaline Earth Metal Oxide Catalysts for Biodiesel Production from Palm Oil: Elucidation of Process Behaviors and Modeling Using Response Surface Methodology. Iran. J. Chem. Chem. Eng. (IJCCE), 32(1): 113-26 (2013)
[27] Zhi W., Song J., Ouyang F., Bi J., Application of Response Surface Methodology to the Modeling of α-Aamylase Purification by Aqueous Two-Phase Systems, J. Biotechnol. 118(2): 157-165 (2005).
[28] Dembczyński R., Białas W., Jankowski T., Partitioning of Lysozyme in Aqueous Two-Phase Systems Containing Ethylene Oxide-Propylene Oxide Copolymer and Potassium Phosphates, Food Bioprod Process, 91(3): 292-302 (2013).
[29] Alcântara L.A.P., do Nascimento K.S., Mourão C.A., Minim V.P.R., Minim L.A., Aqueous Two-Phase Poly (ethylene glycol)–Sodium Polyacrylate System for Amyloglucosidase Purification: Equilibrium Diagrams and Partitioning Studies, Sep. Purif. Technol., 118: 888-894 (2013).
[31] Cheng Z., Cheng L., Song H., Yu L., Zhong F., Shen Q., Hu H., Aqueous Two-Phase System for Preliminary Purification of Lignans from Fruits of Schisandra Chinensis Baill, Sep. Purif. Technol., 166: 16-25 (2016).
[32] Goleij M., Fakhraee H., Response Surface Methodology Optimization of Cobalt (II) and Lead (II) Removal from Aqueous Solution Using MWCNT-Fe3O4 Nanocomposite. Iran. J. Chem. Chem. Eng. (IJCCE), 36(5): 129-141. (2017).
[34] Wang S., Qin L., Zhou Z., Wang J., Solubility and Solution Thermodynamics of Betaine in Different Pure Solvents and Binary Mixtures, J. Chem. Eng. Data, 57(8): 2128-2135 (2012).
[37] Alam M.S., Kumar S., Naqvi A.Z., Study of the Cloud point of an Amphiphilic Antidepressant Drug: Influence of Surfactants, Polymers, and Non-Electrolytes, ‎Colloids Surf., A 287(1): 197-202 (2006).
[38] Alam M.S., Naqvi A.Z., Tuning of the Cloud Point of Promethazine Hydrochloride with Surfactants
and Polymers,
J. Surfact Detergents, 10(1): 35-40 (2007).
[39] Alam M.S., Naqvi A.Z., Study of the Cloud Point of the Phenothiazine Drug Chlorpromazine Hydrochloride: Effect of Surfactants and Polymers, J. Dispersion Sci. Technol., 29(2): 274-279 (2008).
[40] Alam M.S., Mandal A.B., Thermodynamics at the Cloud Point of Phenothiazine Drug Chlorpromazine Hydrochloride− Additive Systems, J. Chem. Eng. Data, 55(4): 1693-1699 (2010).
[41] Mokhtarani B., Mortaheb H.R., Mafi M., Amini M.H., Partitioning of α-Lactalbumin and β-Lactoglobulin in Aqueous Two-Phase Systems of Polyvinylpyrrolidone and Potassium Phosphate, J. Chromatogr. B 879(11): 721-726 (2011).
[42] Tubío G., Pellegrini L., Nerli B.B., Picó G.A., Liquid-Liquid Equilibria of Aqueous Two-Phase Systems Containing Poly (ethylene glycols) of Different Molecular Weight and Sodium Citrate, J. Chem. Eng. Data., 51(1): 209-212 (2006).
[48] de Boeck S., Stockx J., Egg white lLysozyme is the Major Protein of the Hen's Egg Vitelline Membrane, Int. J. Biochem., 18(7): 617-622 (1986).
[50] Bora M., Borthakur S., Rao P., Dutta N., Aqueous Two-Phase Partitioning of Cephalosporin Antibiotics: Effect of Solute Chemical Nature, Sep. Purif. Technol., 45(2): 153-156 (2005).
[52] Alves J.G., Chumpitaz L.D., da Silva L.H., Franco T.T., Meirelles A.J., Partitioning of Whey Proteins, Bovine Serum Albumin and Porcine Insulin in Aqueous Two-Phase Systems, .J Chromatogr. B Biomed. Sci. Appl., 743(1): 235-239 (2000).
[53] Rodríguez-Durán L.V., Spelzini D., Boeris V., Aguilar C.N., Picó G.A., Partition in Aqueous Two-Phase System: Its Application in Downstream Processing of Tannase from Aspergillus Niger, Colloids Surf., B 101: 392-397 (2013).