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) we re covalently linked to bisoxirane-activated nylon membranes, Cibacron Blue F3G-A was immobilized on to these membranes to yield a dye-affin ity membrane, The hydrodynamic permeability of affinity membranes was reduced to approximate to 50% of that of the original Nylon membrane d ue to extension of polymer coils into now-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 mu g/cm(2) for HSA to 316 mu g/c m(2) for MDH, The protein recovery of HSA was 100% on dextran-coated m embranes 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 purific ation factors of 7.4, 8.9 and 11.2 for recombinant alanine dehydrogena se from Mycobacterium tuberculosis for HEC-, dextran- and PVA-coated m embranes respectively, Dynamic capacities decreased remarkably to appr oximate to 3 mu g/cm(2) due to co-adsorption of host proteins, The pre sence of cell debris caused only a slight decrease of purification fac tors, but a dramatic decrease of the permeability of affinity membrane s due to development of a particle layer in front of the membranes, Al though enzyme recoveries were up to 90% using cell-free supernatant, m ore than 50% of the product was lost due to polarization, concentratio n and rejection at particle layers when using crude homogenates, In or der to further improve this integrated downstream process, sophisticat ed 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.