Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase.
A whole broth extraction using an aqueous two-phase system (ATPS) composed by 5% (w/w) PEG 3350 and 15% (w/w) phosphate was used for the scale-up extraction and isolation of a recombinant Fusarium solani pisi cutinase, an extracellular mutant enzyme expressed in Saccharomyces cerevisiae, containing a fusion peptide (WP)4. The experiments were carried out at three different scales (10 ml, 1 l and 30 l). Mixing time and stirrer speed were evaluated at lab scale (1 l) with two different system compositions. Stirrer speed between 400 and 800 rpm and mixing time between 2 and 5 min led to the highest recoveries of cutinase. In all cases, inclusive of pilot scale (30 l), the equilibrium was reached after a few minutes. The performance of ATPS was reproducible within the scale range of 0.010-30 l and provided a standard deviation of the yield lower than 8%, leading to (i) a partition coefficient over 50, (ii) a yield over 95% and (iii) a concentration factor over 5. The fusion of the peptide (WP)4 to the cutinase protein enabled a 400 increase of the partition coefficient relative to the wild-type strain.
Costa
M J
MJ
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisboa, Portugal.
Cunha
M T
MT
Cabral
J M
JM
Aires-Barros
M R
MR
D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D005670:Fusarium / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D010865:Pilot Projects; D011092:Polyethylene Glycols; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014867:Water
D011993:Recombinant Fusion Proteins; D014867:Water; D011092:Polyethylene Glycols; D002265:Carboxylic Ester Hydrolases; C019952:cutinase