Poly(hydroxyethylmethacrylate-N-methacryloyl-(L)-histidine-methyl-ester) based metal-chelate affinity adsorbent for separation of lysozyme


Senel S., Elmas B., Camli T., Andac M., Denizli A.

SEPARATION SCIENCE AND TECHNOLOGY, vol.39, no.16, pp.3783-3795, 2004 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 39 Issue: 16
  • Publication Date: 2004
  • Doi Number: 10.1081/ss-200041105
  • Journal Name: SEPARATION SCIENCE AND TECHNOLOGY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.3783-3795
  • Keywords: affinity chromatography, lysozyme separation, metal-chelate affinity beads, histidine, poly(HEMA), IMMUNOGLOBULIN-G, HUMAN PLASMA, CHROMATOGRAPHY, PURIFICATION, PROTEINS, ULTRAFILTRATION, MEMBRANES, PEPTIDES
  • Hacettepe University Affiliated: Yes

Abstract

Comonomer and/or metal-chelating ligand N-methacryloyl-(L)-histidine-methylester (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. Spherical beads with an average diameter of 75-125 mum were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Poly(HEMA-MAH) beads had a specific surface area of 18.3 m(2)/g. Elemental analysis of MAH for nitrogen was estimated as 895 mumol/g of polymer. Then the beads were loaded with different metal ions (i.e. Zn2+, Cu2+, Ni2+) to form the metal chelate. The effect of pH, concentration of lysozyme, and metal type on the adsorption of lysozyme to the metal-chelated beads was examined in a batch reactor. Purification of lysozyme from egg-white was also investigated. Maximum lysozyme adsorption capacity of poly(HEMA-MAH) beads was found to be 8.7 mg/g at pH 7.0 in phosphate buffer. Lysozyme adsorption capacity of Zn2+, Cu2+, and Ni2+-chelated beads was higher than that of non-chelated beads. The maximum capacities of Ni2+, Zn2+, or Cu2+-chelated beads were 11.5, 12.6, and 37.1 mg/g, respectively. A significant amount of the adsorbed lysozyme (up to 97%) was eluted in I h in the elution medium containing 25 mM EDTA at pH 4.9. Repeated adsorption-desorption process showed that this novel metal chelated beads are suitable for lysozyme adsorption. Purification of lysozyme was monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 80% with recovery about 75%.