Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth.
Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.
Charoenrat
Theppanya
T
School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
Ketudat-Cairns
Mariena
M
Jahic
Mehmedalija
M
Enfors
Sven-Olof
SO
Veide
Andres
A
D000327:Adsorption; D018929:Cell Culture Techniques / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D002845:Chromatography / Q000379:methods*; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014462:Ultrafiltration / Q000379:methods*; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D001617:beta-Glucosidase