Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: A comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

• Published in: Journal of biotechnology 2005-10, Vol.119 (4), p.436-448
• Main Authors: Ling, Tau Chuan, Lyddiatt, Andrew
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Bioreactors - microbiology; Biotechnology; Cell Culture Techniques - methods; Cell disruption; Chromatography, Liquid - methods; Coloring Agents; Dye-ligand; Fluidised bed; Fundamental and applied biological sciences. Psychology; G3PDH; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - isolation & purification; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - metabolism; Intracellular proteins; Ligands; Microfluidics - methods; Physical Stimulation - methods; Polymers; Saccharomyces cerevisiae; Saccharomyces cerevisiae - isolation & purification; Saccharomyces cerevisiae - metabolism; Systems Integration
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2005.05.029

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; ρ = 2.65 g ml −1) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 l h −1) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2 IU mg −1, after a single step elution with 0.15 M NaCl. The generic application of this approach has been evaluated.