Purificaiton of alcohol dehydrogenase from Saccharomyces cerevisiae using magnetic dye-ligand affinity nanostructures

Reactive dyes are used as affinity ligands for protein purification since they may interact with the active sites by mimicking the structure of the substrates and coenzymes of enzymes. In this thesis, dye ligand affinity chromatography was used for purification of alcohol dehydrogenase from Saccharomyces cerevisiae (S. cerevisiae) which uses NAD+ as coenzyme. For this purpose poly(2-hydroxyethyl methacrylate) [poly(HEMA)] nanostructures were synthesized and Reactive Green 19 was covalently attached to nanostructures as dye ligand by nucleophilic substitution reaction. Characterization of these nanostructures was performed using FTIR, ESR, SEM, AFM and EDX analysis. Particles synthesized were found to be spherical in shape with approximately 70 nm diameter and magnetic. Adsorption of alcohol dehydrogenase on the dye attached nanostructures was performed in batch system and the effects of pH, alcohol dehydrogenase concentration, temperature and ionic strength on adsorption were investigated. Maximum alcohol dehydrogenase adsorption on dye attached nanostructures was measured to be 176,09 mg/g polymer at 0,5 mg/mL of alcohol dehydrogenase concentration and phosphate buffer at pH 7,0. Desorption of adsorbed alcohol dehydrogenase on the nanostructures was carried out by 1,0 M NaCl solution and desorption rate was calculated to be 98,4 %. Synthesized nanostructures were used for purification of alcohol dehydrogenase from S. cerevisiae following optimization of adsorption and desorption conditions of alcohol dehydrogenase from aqueous solutions. At the end of purification process, alcohol dehydrogenase was purified with 71,26 % recovery and 45,63 fold purification with a single step. Purity of enzyme was demonstrated with silver stained SDS-PAGE.