Purification of Lysozyme with Congo Red Immobilized Cryogel Discs

Abstract

In this study, congo red immobilized poly hydroxyethyl methacrylate [CR-PHEMA] cryogel discs were pre- pared for purification of lysozyme. CR-PHEMA cryogel discs were characterized by swelling test, elemental analysis and scanning electron microscopy. Lysozyme adsorption was performed from aqueous solutions and maximum lysozyme adsorption capacity of CR-PHEMA cryogel discs was found to be 136.6 mg/g. Effects of initial lysozyme concentration, pH and ionic strength on lysozyme adsorption capacity of CR-PHEMA cryogel discs were investigated. Maximum adsorption was obtained at pH 8.0. In the last step of this study, lysozyme was purified from egg white using CR-PHEMA cryogel discs. Egg white fractions were observed on sodium do- decyl sulfate polyacrylamide gel electrophoresis.

Keywords

• Congo red,lysozyme,PHEMA,cryogel

Kongo Kırmızısı İmmobilize Kriyojel Disklerle Lizozim Saflaştırılması

Abstract

B u çalışmada, lizozim saflaştırılması için congo kırmızısı immobilize edilmiş poli hidroksietil metakrilat [CRPHEMA] kriyojel diskler hazırlanmıştır. CR-PHEMA kriyojel diskler şişme testi, elementel analiz ve taramalı elektron mikroskobu ile karakterize edilmiştir. Lizozim adsorpsiyonu sulu çözeltilerden gerçekleştirilmiş ve CRPHEMA kriyojel disklerin maksimum lizozim adsorpsiyon kapasitesi 136.6 mg/g olarak bulunmuştur. Başlangıç lizozim derişiminin, pH’ın ve iyonik şiddetin CR-PHEMA kriyojel disklerin lizozim adsorpsiyon kapasitesine olan etkileri araştırılmıştır. Maksimum adsorpsiyon pH 8.0’de elde edilmiştir. Bu çalışmanın son basamağında, lizozim CR-PHEMA kriyojel diskler kullanılarak yumurta beyazından saflaştırılmıştır. Yumurta beyazı fraksiyonları sodyum dodesil sülfat poliakrilamid jel elektroforezinde incelenmiştir

Keywords

• Congo kırmızısı,lizozim,PHEMA,kriyojel

References

1. 1. A. Denizli, B. Salih, A. Kozluca, E. Pişkin, Comparison of albumin binding capacities of three different reactive dye-derivatized poly(ethylene glycol dimethacrylatehydroxyethyl methacrylate) microbeads, J. Biomater. Sci. Polym. Ed., 8 (1997) 411.
2. 2. A. Denizli, E. Pişkin, Dye-ligand affinity systems, J. Biochem. Biophys. Methods, 49 (2001) 391.
3. 3. Z. Li, M. Cao, W. Zhang, L. Liu, J. Wang, W. Ge, Y. Yuan, T. Yue, R. Li, W.W. Yu, Affinity adsorption of lysozyme with Reactive Red 120 modified magnetic chitosan microspheres, Food Chem., 145 (2014) 749.
4. 4. F.M. Plieva, I.Y. Galaev, B. Mattiasson, Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulatecontaining fluids and cell culture applications, J Sep Sci., 30 (2007) 1657.
5. 5. V.I. Lozinsky, I.Y. Galaev, F.M. Plieva, I.N. Savina, H. Jungvid, B. Mattiasson, Polymeric cryogels as promising materials of biotechnological interest, Trends Biotechnol., 21 (2003) 445.
6. 6. V.I. Lozinsky, F.M. Plieva, I.Y. Galaev, Bo Mattiasson, The potential of polymeric cryogels in bioseparation, Bioseparation 10 (2002) 163.
7. 7. Kumar, A. Srivastava, Cell separation using cryogelbased affinity chromatography, Nature Protocols, 5 (2010) 1737.
8. 8. L. Callewaert, J.M. Van Herreweghe, L. Vanderkelen, S. Leysen, A. Voet, C.W. Michiels, Guards of the great wall: bacterial lysozyme inhibitors, Trends Microbiol., 20 (2012) 501.

9. 9. N. Schneider, K. Werkmeister, C.M. Becker, M. Pischetsrieder, Prevalence and stability of lysozyme in cheese, Food Chemistry, 128 (2011) 145.
10. 10. L. Callewaert, M. Walmagh, C.W. Michiels, R. Lavigne, Food applications of bacterial cell wall hydrolases, Curr Opin Biotechnol., 22 (2011) 164.
11. 11. H.M. Chang, C.C. Yang, Y.C. Chang, Rapid separation of lysozyme from chicken egg white by reductants and thermal treatment, J. Agr. Food Chem., 48 (2000) 161.
12. 12. P. Arvidsson, F.M. Plieva, I.N. Savina, V.I. Lozinsky, S. Fexby, L. Bülow, I.Y. Galaev, B. Mattiasson, Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns, J. Chromatogr. A, 977 (2002) 27.
13. 13. H. Alkan, N. Bereli, Z. Baysal, A. Denizli, Antibody purification with protein A attached supermacroporous poly(hydroxyethyl methacrylate) cryogel, Biochem. Eng. J., 45 (2009) 201.
14. 14. M. Odabasi, A. Denizli, Cibacron Blue F3GA-attached magnetic poly(2-hydroxyethyl methacrylate) beads for human serum albumin adsorption, Polym. Int., 53 (2004) 332.
15. 15. M. van der Veen, W. Norde, M.C. Stuart, Electrostatic interactions in protein adsorption probed by comparing lysozyme and succinylated lysozyme, Colloid Surface B., 35 (2004) 33.