Purification of yeast alcohol dehyrogenase by immobilized metal affinity chromatography

PURIFICATION OF ALCOHOL DEHYDROGENASE BY IMMOBIKIZED METAL AFFINITY CHROMATOGRAPHY Begüm Akduman M. Sc. Thesis, Department of Chemistry Supervisor: Assoc. Prof. Dr. Deniz AKTAŞ UYGUN 2013, 76 Pages In this thesis, poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a fonctional comonomer and N,N'-methylene-bisacrylamide(MBAAm) as a crosslinker. Iminodiacetic acid (IDA) fonctional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn2+ ions were chealeted with this structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 µm size. The equilibrium swelling degree of Zn2+ chealeted poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn2+ chealeted poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, ionic strengt hand flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption wasd etermined to be 9,94 mg/g cryogel at 1,0 mg/mL alcohol dehyrogenase concentration and acetate buffer at pH 5,0 with flow rate of 0,5 mL/min. Desorption of adsorbed alcohol dehyrogenase was carried out by 1,0 M NaCI at pH 8,0 phosphate buffer and desorption rate was found to be 93,5 %. Additionally, these cryogels was used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates. Keywords: Alcoholdehydrogenase, yeast, cryogel, IMAC, iminodiaceticacid