A study of combined filtration and adsorption on nylon-based dye-affinity membranes: separation of recombinant L-alanine dehydrogenase from crude fermentation broth.
Dextran, hydroxyethylcellulose (HEC), and poly(vinyl alcohol) PVA were covalently linked to bisoxirane-activated nylon membranes. Cibacron Blue F3G-A was immobilized on to these membranes to yield a dye-affinity membrane. The hydrodynamic permeability of affinity membranes was reduced to approximately 50% of that of the original Nylon membrane due to extension of polymer coils into flow-through pores. Adsorption of pre-purified human serum albumin (HSA) and malate dehydrogenase (MDH) displayed highest maximum binding capacities on HEC-coated dye-ligand-affinity membranes, ranging from (163 micrograms/cm2 for HSA to 316 micrograms/cm2 for MDH. The protein recovery of HSA was 100% on dextran-coated membranes compared with 70% on PVA-coated membranes, whereas almost 100% recovery was found for MDH, independent of the polymer. Application of crude supernatant from recombinant Escherichia coli yielded purification factors of 7.4, 8.9 and 11.2 for recombinant alanine dehydrogenase from Mycobacterium tuberculosis for HEC-, dextran- and PVA-coated membranes respectively. Dynamic capacities decreased remarkably to approximately 3 micrograms/cm2 due to co-adsorption of host proteins. The presence of cell debris caused only a slight decrease of purification factors, but a dramatic decrease of the permeability of affinity membranes due to development of a particle layer in front of the membranes. Although enzyme recoveries were up to 90% using cell-free supernatant, more than 50% of the product was lost due to polarization, concentration and rejection at particle layers when using crude homogenates. In order to further improve this integrated downstream process, sophisticated membrane techniques are required by which the formation of a filter cake is circumvented. Further refinement of polymer-coated membranes would not help one to avoid this problem.
Weissenborn
M
M
Biochemical Engineering Division, Gesellschaft für Biotechnologische Forschung m.b.H., Braunschweig, Federal Republic of Germany.
Hutter
B
B
Singh
M
M
Beeskow
T C
TC
Anspach
F B
FB
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D005285:Fermentation; D005374:Filtration; D008291:Malate Dehydrogenase / Q000378:metabolism; D008567:Membranes, Artificial; D008855:Microscopy, Electron, Scanning; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D012709:Serum Albumin / Q000378:metabolism; D013816:Thermodynamics
D008567:Membranes, Artificial; D011994:Recombinant Proteins; D012709:Serum Albumin; D008291:Malate Dehydrogenase; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase