title: Development of an affinity cryogel for one step purification of lysozyme from chicken egg white.
abstract: In this study, a supermacroporous polyacrylamide cryogel was produced by cryo-polymerization and activated with Tris(hydroxymethyl)aminomethane (Tris-cryogel) to be applied as an affinity ligand for a one step purification of lysozyme (LYZ), directly from chicken egg white (EW). The Tris-cryogel presented interconnected pores with size varying in the range of 20-80μm and swelling capacity of 19.6±0.9g/g. The axial dispersion of the Tris-cryogel was analyzed at different flow velocities and mobile phase viscosities. It was verified that higher viscosity resulted in a higher degree of dispersion, causing the HETP values to increase from 0.04cm to 0.8cm. Adsorption isotherms were measured at 15°C and 35°C at pH 7.5. A Langmuir model was fitted to the equilibrium data, with a maximum adsorptive capacity of 285mg/g at 15°C and 363mg/g at 35°C. Thermodynamic analysis based on the Van't Hoff relationship showed that the process was spontaneous and enthalpically driven. Lysozyme was purified directly from egg white in a one step purification process at different pH values (7.5, 8.5 and 9.5). Independent of the pH, the specificity of Tris-cryogel for lysozyme adsorption was confirmed. At pH 7.5, yield and purification fold were higher (30% and 45). In addition, the effect of the dilution rate on egg white and flow velocity were also analyzed and it was shown that flow velocity did not affected purification and column efficiency, and that diluting the egg white increased yield to 70% with a purification fold of 23. Results show Tris-cryogel is a promising matrix for use in high throughput purification of lysozyme from egg white.
authors.affiliation: Department of Food Engineering and Technology, UNESP- São Paulo State University, 15054-000, São José do Rio Preto, SP, Brazil.
authors.forename: Paula Chequer Gouveia
authors.initials: PCG
authors.lastname: Mól
authors.affiliation: Department of Food Science, Federal University of Lavras, 37200-000, Lavras, MG, Brazil.
authors.forename: Lizzy Ayra Alcântara
authors.initials: LAA
authors.lastname: Veríssimo
authors.affiliation: Department of Food Technology, Federal University of Viçosa, 36570-000, Viçosa, MG, Brazil.
authors.forename: Monique Renon
authors.initials: MR
authors.lastname: Eller
authors.affiliation: Department of Food Technology, Federal University of Viçosa, 36570-000, Viçosa, MG, Brazil.
authors.forename: Valéria Paula Rodrigues
authors.initials: VPR
authors.lastname: Minim
authors.affiliation: Department of Food Technology, Federal University of Viçosa, 36570-000, Viçosa, MG, Brazil. Electronic address: lminim@ufv.br.
authors.forename: Luis Antonio
authors.initials: LA
authors.lastname: Minim
mesh_terms: D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D059985:Cryogels / Q000737:chemistry*; D004529:Egg White / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D009113:Muramidase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D059985:Cryogels; D009113:Muramidase
keywords: Affinity chromatography; Cryogel; Lysozyme; Purification