Purification of glucose oxidase and β-galactosidase by partitioning in a PEG-salt aqueous two-phase system in the presence of PEG-derivatives

Purification of glucose oxidase from Aspergillus niger and that of beta- galactosidase from Kluyveromyces lactis have been attempted using poly(ethylene glycol) (PEG)-sodium sulfate aqueous two phase system (ATPS) in the presence of PEG-derivatives, i.e. PEG-Coomassie brilliant blue G- 250 and PEG-benzoate, PEG-palmitate and PEG-TMA, respectively. The enzymes showed poor partitioning towards the PEG phase in comparison with other proteins in ATPS containing no ligands. Selective partitioning of other proteins was observed towards the PEG phase in the presence of PEG- benzoate and PEG-palmitate enriching beta-galactosidase in the salt phase whereas in the case of glucose oxidase, PEG-Coomassie brilliant blue G-250 derivative worked as a better affinity ligand for other proteins. A 19-fold purification was obtained with the PEG dye derivative after 5 stage cross extractions with 80% recovery of glucose oxidase and an enrichment factor upto similar to 7 for beta-galactosidase with the PEG-TMA derivative. The interaction of PEG-benzoate and PEG-TMA ligands with the active site of beta-galactosidase has been evaluated by molecular modeling. The effect of the molecular weight of glucose oxidase on its partitioning was confirmed as the molecular simulation shows strong affinity interaction of PEG-glucoside with the enzyme.