Direct enzyme adsorption from an unclarified microbial feedstock using suspended bed chromatography.
Suspended bed chromatography (SBC) is a new hybrid technique concomitantly benefiting from batch adsorption, the process advantages of an enclosed system, and its compatibility with established commercial chromatographic contactors and adsorbents. SBC was evaluated in the anion-exchange capture and chromatographic fractionation of native glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from the complex mixture of molecular and particulate moieties that constitute wet-milled bakers' yeast. Method scouting established operating conditions exploiting Whatman Express-Ion Exchanger Q at pH 7.5 and a disrupted biomass concentration equivalent to 3.5% (wet mass/v original intact cells). Partially purified G3PDH was recovered directly from the yeast disruptate in a scaled-down process developed at 1/756 process scale. This was used to establish operating parameters to facilitate process scale-up to exploit a 44 cm I.D. Millipore IsoPak column, 18 kg (swollen mass) of Express-Ion Q anion-exchange cellulose and 275 1 of 3.5% (wet w/v) bakers' yeast disruptate. The generic utility of SBC was demonstrated for direct product adsorption from feedstocks characterised by a modest content of bioparticulates (equivalent to < 4% (wet w/v) disrupted cells). Analyses illustrated an enrichment of G3PDH in respect of enzyme concentration and significant reduction in product turbidity and Pico-Green reactivity (correlated with double stranded (ds) DNA content). The application niche for this new approach to primary protein recovery is discussed with particular reference to the downstream processing of coarsely clarified whole broths, cell disruptates and biological extracts.
Ling
Tau Chuan
TC
University of Birmingham, Biochemical Recovery Group, Centre for Formulation Engineering, Department of Chemical Engineering, Edgbaston, Birmingham B15 2TT, UK.
Lyddiatt
Andrew
A
Carmichael
Ian
I
Purdom
Geoff
G
Hathi
Prit
P
Levison
Peter R
PR
D000327:Adsorption; D018533:Biomass; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry*
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases