Interfacing Pichia pastoris cultivation with expanded bed adsorption.
For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L x h.
Jahic
Mehmedalija
M
Department of Biotechnology, Royal Institute of Technology, KTH, Alba Nova University Center, Roslagstullsbacken 21, S-106 91, Stockholm, Sweden.
Knoblechner
Josef
J
Charoenrat
Theppanya
T
Enfors
Sven-Olof
SO
Veide
Andres
A
D018533:Biomass; D019149:Bioreactors; D002455:Cell Division / Q000187:drug effects; D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods; D003470:Culture Media / Q000494:pharmacology; D005656:Fungal Proteins; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D009172:Mycology / Q000379:methods; D020088:Neocallimastix / Q000201:enzymology / Q000235:genetics; D009994:Osmolar Concentration; D010843:Pichia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
D003470:Culture Media; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica; D002480:Cellulase