Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures.
Direct product sequestration of extracellular proteins from microbial batch cultures can be achieved by continuous or intermittent broth recycle through an external extractive loop. Here, we describe the development of a fluidisable, mixed mode adsorbent, designed to tolerate increasing ionic strength (synonymous with extended productive batch cultures). This facilitated operations for the integrated recovery of an extracellular acid protease from cultures of Yarrowia lipolytica. Mixed mode adsorbents were prepared using chemistries containing hydrophobic and ionic groups. Matrix hydrophobicity and titration ranges were matched to the requirements of integrated protease adsorption. A single expanded bed was able to service the productive phase of growth without recourse to the pH adjustment of the broth previously required for ion exchange adsorption. This resulted in increased yields of product, accompanied by further increases in enzyme specific activity. A step change from pH 4.5 to 2.6, across the isoelectric point of the protease, enabled high resolution fixed bed elution induced by electrostatic repulsion. The generic application of mixed mode chemistries, which combine the physical robustness of ion-exchange ligands in sanitisation and sterilisation procedures with a selectivity, which approaches that of affinity interactions, is discussed.
Hamilton
G E
GE
Biochemical Recovery Group, Centre for Bioprocess Engineering, School of Chemical Engineering, University of Birmingham, Birmingham, UK.
Luechau
F
F
Burton
S C
SC
Lyddiatt
A
A
D000327:Adsorption; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005656:Fungal Proteins; D006863:Hydrogen-Ion Concentration; D015003:Yeasts / Q000201:enzymology*
D005656:Fungal Proteins; C103006:AXP protein, Yarrowia lipolytica; D016282:Aspartic Acid Endopeptidases