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.
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; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-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 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.
Ling
Tau Chuan
TC
Biochemical Recovery Group, Centre for Formulation Engineering, Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. ltc555@eng.upm.edu.my
Lyddiatt
Andrew
A
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004396:Coloring Agents; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000302:isolation & purification* / Q000378:metabolism*; D008024:Ligands; D044085:Microfluidics / Q000379:methods; D010812:Physical Stimulation / Q000379:methods; D011108:Polymers; D012441:Saccharomyces cerevisiae / Q000302:isolation & purification* / Q000378:metabolism*; D018511:Systems Integration
D004396:Coloring Agents; D008024:Ligands; D011108:Polymers; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)