Abstract
A fast and efficient purification method for recombinant glucose oxidase (rGOx) for flask fermentation scale (up to 2 L) was designed for the purposes of characterization of rGOx mutants during directed protein evolution. The Aspergillus niger GOx was cloned into a pYES2-αMF-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.
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