Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins.
Genetic engineering was integrated with the production and purification of Fusarium solani pisi cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were cutinase wild type and two fusion proteins of cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)(2) and (WP)(4). The (WP)(4) fusion protein enabled a 300-fold increase of the cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the cutinase-(WP)(2) proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)(4) fusion proteins, respectively.
Cunha
M T
MT
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, Portugal.
Costa
M J L
MJ
Calado
C R C
CR
Fonseca
L P
LP
Aires-Barros
M R
MR
Cabral
J M S
JM
D019149:Bioreactors; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005110:Extracellular Space / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005670:Fusarium / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D018511:Systems Integration
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase