Benchmarking polymeric cryogels for immobilized metal affinity chromatography

Cryogels are polymers prepared in frozen environment and seen as new separation matrices with their applications in many bioseparation methods. They have significant benefits including supermacroporosity, short diffusion path, low pressure, and low resistance to both adsorption and elution. Macro and connected pores give cryogels a unique spongy structure. Immobilized metal affinity chromatography (IMAC) is a generally employed analytical separation method for the purification of biomolecules. Several transition ions create stable complexes with electron-rich compounds. IMAC sorbent is obtained by complexing first-order transition metal ions over chelating agents. Lysozyme is an enzyme found in various vertebrate cells and secretions. Common applications include its use as a cell disrupting agent, as an anti-bacterial agent, as a food additive, and as a medicine for the treatment of infections and ulcers. In this study, cryogel-based polymeric material prepared by free-radical polymerization method with hydroxyethyl methacrylate/glycidyl methacrylate monomer pair were covalently interacted with iminodiacetic acid metal chelating agent, and regions showing affinity for lysozyme enzyme were formed by binding with Ni(II) ions. The cryogel was first characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, thermal gravimetric analysis, X-ray photoelectron spectroscopy and swelling test. Then, the effects of pH, lysozyme concentration, temperature, ionic strength and flow rate on lysozyme adsorption capacity were explored and optimum conditions were found.

Keywords:

Enzyme adsorption, immobilized metal affinity chromatograpy cryogel.,

PDF

___

1. J. Thömmes, M. Etzel, Alternatives to chromatographic separations, Biotechnol. Prog., 23(1) (2007) 42-45.
2. Y. Saylan, A. Denizli, Supermacroporous composite cryogels in biomedical applications, Gels, 5(2) (2019) 20
3. M. Andaç, I.Y. Galaev, A. Denizli, Affinity based and molecularly imprinted cryogels: Applications in biomacromolecule purification, J. Chromatogr. B, 1021 (2016) 69-80.
4. V.I. Lozinsky, Cryostructuring of polymeric systems. 50.† cryogels and cryotropic gel-formation: Terms and definitions. Gels, 4 (2018) 77-89.
5. S. Akgönüllü, H. Yavuz, A. Denizli, Preparation of imprinted cryogel cartridge for chiral separation of l-phenylalanine, Artif. Cell. Nanomed. Biotechnol., 45 (2017) 800-807.
6. G.C. Ingavle, L.W.J. Baillie, Y. Zheng, E.K. Lis, I.N. Savina, C.A. Howell, S.V. Mikhalovsky, S.R. Sandeman, Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen, Biomaterials, 50 (2015) 140-153.
7. K. Çetin, A. Denizli, 5-Fluorouracil delivery from metal-ion mediated molecularly imprinted cryogel discs, Coll. Surf. B, 126 (2015) 401-406.
8. C.Y. Kuo, C.H. Chen, C.Y. Hsiao, J.P. Chen, Incorporation of chitosan in biomimetic gelatin/chondroitin-6-sulfate/hyaluronan cryogel for cartilage tissue engineering, Carbohydr. Polym., 117 (2015) 722-730.
9. N.K. Singh, R.N. Dsouza, M. Grasselli, M. Fernández-Lahore, High capacity cryogel-type adsorbents for protein purification, J. Chromatogr. A, 1355 (2014) 143-148.
10. J. Porath, Immobilized metal ion affinity chromatography, Protein Expr. Purif., 3(4) (1992) 263-281.
11. E.K.M. Ueda, P.W. Gout, L. Morganti, Current and prospective applications of metal ion-protein binding, J. Chromatogr. A, 988(1) (2003) 1-23.
12. N. Tüzmen, T. Kalburcu, A. Denizli, Immobilization of catalase via adsorption onto metal-chelated affinity cryogels, Process Biochem. 47(1) (2012) 26-33.
13. S. Mallik, R.D. Johnson, F.H. Arnold, Selective recognition of bis-imidazoles by complementary bis-metal ion complexes, JACS, 115 (1993) 2518-2520.
14. A. Denizli, Purification of antibodies by affinity chromatography, Hacettepe J. Biol. Chem., 39(1) (2011) 1-18.
15. E. Tamahkar, A. Denizli, Metal ion coordination interactions for biomolecule recognition: a review, Hittite J. Sci. Eng., 1(1) (2014) 21-26.
16. N. Bereli, Y. Saylan, L. Uzun, R. Say, A. Denizli, L-histidine imprinted supermacroporous cryogels for protein recognition, Sep. Purif. Technol., 82 (2011) 28-35.
17. Y. Saylan, E. Tamahkar, A. Denizli, Recognition of lysozyme using surface imprinted bacterial cellulose nanofibers, J. Biomater. Sci. Polym. Ed., 28(16) (2017) 1950-1965.
18. A. Vasilescu, Q. Wang, M. Li, R. Boukherroub, S. Szunerits, Aptamer-based electrochemical sensing of lysozyme, Chemosensors, 4 (2016) 10-30.
19. Y. Saylan, F. Yılmaz, A. Derazshamshir, E. Yılmaz, A. Denizli, Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor, J. Mol. Recog., 30(9) (2017) e2631.
20. C. Ocana, A. Hayat, R.K. Mishra, A. Vasilescu, M. del Valle, J.L. Marty, Label free aptasensor for lysozyme detection: a comparison of the analytical performance of two aptamers. Bioelectrochem., 105 (2015) 72-77.
21. G. Öztürk, Y. Saylan, A. Denizli, Designing composite cryogel carriers for tyrosine adsorption. Sep. Purif. Technol., 254 (2021) 117622.
22. A. Baimenov, D. Berillo, S. Azat, T. Nurgozhin, V. Inglezakis, Removal of Cd2+ from water by use of super-macroporous cryogels and comparison to commercial adsorbents. Polymers, 12(10) (2020) 2405.
23. B. Wan, J. Li, F. Ma, N. Yu, W. Zhang, L. Jiang, H. Wei, Preparation and properties of cryogel based on poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate). Langmuir, 35(9) (2019) 3284-3294.
24. R.H. Şenay, S.M Gökalp, E. Türker, E. Feyzioğlu, A. Aslan, S. Akgöl, A new morphological approach for removing acid dye from leather waste water: Preparation and characterization of metalchelated spherical particulated membranes (SPMs). J. Environ. Manage., 151 (2015) 295-302.
25. E. Yeşilova, B. Osman, A. Kara, E. Tümay Özer, Molecularly imprinted particle embedded composite cryogel for selective tetracycline adsorption. Sep. Purif. Technol., 200 (2018) 155-163.
26. T. Matsunaga, T. Hishiya, T. Takeuchi. Surface plasmon resonance sensor for lysozyme based on molecularly imprinted thin films. Anal Chim Acta., 591 (2007) 63-67.
27. S. Öztürk, N. Demir, Development of a novel IMAC sorbent for the identification of melamine in dairy products by HPLC. J. Food Compos. Anal., 100 (2021) 103931.
28. I. Göktürk, I. Perçin, A. Denizli, Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs. Prep. Biochem. Biotechnol., 46(6) (2016) 602-609.
29. Y. Saylan, N. Bereli, L. Uzun, A. Denizli, Monolithic boronate affinity columns for IgG separation. Sep. Sci. Technol., 49(10) (2014) 1555-1565.
30. Y. Saylan, L. Uzun, A. Denizli, Alanine functionalized magnetic nanoparticles for reversible amyloglucosidase immobilization. Ind. Eng. Chem. Res., 54(1) (2015) 454-461.
31. F. Inci, Y. Saylan, A.M. Kojouri, M.G. Ogut, A. Denizli, U. Demirci, A disposable microfluidic-integrated hand-held plasmonic platform for protein detection. Appl. Mat. Today, 18 (2020) 100478.
32. M. Odabaşı, L. Uzun, A. Denizli, Porous magnetic chelator support for albumin adsorption by ımmobilized metal affinity separation. J. Appl. Polym. Sci., 93 (2004) 2501-2510.
33. S. Emir, R. Say, H. Yavuz, A. Denizli, A new metal chelate affinity adsorbent for cytochrome c. Biotechnol. Prog., 20 (2004) 223-228.
34. E.B. Altıntaş, N. Tüzmen, L. Uzun, A. Denizli, Immobilized metal affinity adsorption for antibody depletion from human serum with monosize beads. Ind. Eng. Chem. Res., 46 (2007) 7802-7810.
35. N. Bereli, G. Şener, E. B. Altıntaş, H. Yavuz, A. Denizli, Poly(glycidyl methacrylate) beads embedded cryogels for pseudo-specific affinity depletion of albumin and immunoglobulin G. Mat. Sci. Eng. C, 30 (2010) 323-329.
36. E.B. Altıntas, D. Türkmen, V. Karakoç, A. Denizli, Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads. Coll. Surf. B Bioint., 85 (2011) 235-240.
37. M. Uygun, B. Akduman, S. Akgöl, A. Denizli, A new metal-chelated cryogel for reversible ımmobilization of urease. Appl. Biochem. Biotechnol., 170 (2013) 1815-1826.
38. B. Akduman, M. Uygun, D. Aktaş Uygun, S. Akgöl, A. Denizli, Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels. Mat. Sci. Eng. C, 33(8) (2013) 4842-4848.
39. F. Kartal, A. Denizli, Surface molecularly imprinted magnetic microspheres for the recognition of albumin. J. Sep. Sci., 37 (2014) 2077-2086.
40. D. Aktaş Uygun, M. Uygun, S. Akgöl, A. Denizli, Reversible adsorption of catalase onto Fe3+ chelated poly(AAm-GMA)-IDA cryogels. Mat. Sci. Eng. C, 50 (2015) 379-385.