Purification of glucose oxidase from complex fermentation medium using tandem chromatography

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Recombinant glucose oxidase (rGOx) is crucial for protein evolution studies.
• Efficient purification methods are needed for characterizing rGOx mutants.
• Current methods may require extensive sample preparation.

Purpose of the Study:

• To develop a fast and efficient purification method for rGOx.
• To enable characterization of rGOx mutants from flask fermentation.
• To streamline purification from complex yeast expression media.

Main Methods:

• Designed a Hydrophobic Interaction Chromatography (HIC)/Size Exclusion Chromatography (SEC) tandem system.
• Expressed Aspergillus niger GOx extracellularly in Saccharomyces cerevisiae.
• Utilized Butyl 650s for HIC and Sephadex 200 for SEC.

Main Results:

• Successfully purified rGOx from conditioned complex expression medium.
• Minimized sample preparation steps, requiring only conductivity, pH, and coarse filtering adjustments.
• Achieved high purity confirmed by electrophoretic and UV-vis spectrophotometric analyses.

Conclusions:

• The developed HIC/SEC method is effective for rapid rGOx purification.
• This method facilitates efficient characterization of rGOx mutants.
• The streamlined process is suitable for flask fermentation scale.