Purification of glucose oxidase from complex fermentation medium using tandem chromatography.

A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-alphaMF-GOx construct and expressed extracellularly in yeast Saccharomyces cerevisiae. Hydrophobic interaction (HIC)/size exclusion (SEC)-tandem chromatographic system was designed for direct purification of rGOx from a conditioned complex expression medium with minimum preceding sample preparation (only adjustments to conductivity, pH and coarse filtering). HIC on Butyl 650s (50 mM ammonium acetate pH 5.5 and 1.5 M ammonium sulphate) absorbs GOx from the medium and later it is eluted by 100% stepwise gradient with salt free buffer directly into SEC column (Sephadex 200) for desalting and final polishing separation. The electrophoretic and UV-vis spectrophotometric analyses have proven enzyme purity after purification.