Partition features and renaturation enhancement of chymosin in aqueous two-phase systems.
Aqueous two-phase systems of polyethylene glycol (molecular mass 1450, 3350 and 6000)-phosphate and polyethylene-polypropylene oxide (molecular mass 8400)-maltodextrin systems were used in order to study the partition features of recombinant chymosin from inclusion bodies. These systems in the presence of 8M urea were used for the solubilization of inclusion bodies containing recombinant chymosin and for the oxidative renaturation of this protein. Recombinant chymosin showed to be partitioned in favour of the top phase in all studied systems with a partition coefficient between 4 and 6. The recovery of the chymosin biological activity was 32% in the polyethylene-polypropylene oxide, while in the polyethylene glycol-phosphate the recovery was 50-59%. The results indicate that the liquid-liquid extraction would be an adequate tool able to isolate and concentrate chymosin from inclusion bodies with a yield of biological activity higher than that obtained from the standard method (43%).
Reh
Georgina
G
Physical Chemistry Department, Faculty of Biochemical and Pharmaceutical Sciences, CONICET and FonCyT, National University of Rosario (S2002RLK), Rosario, Argentina.
Spelzini
Dario
D
Tubío
Gisela
G
Picó
Guillermo
G
Farruggia
Beatriz
B
D000818:Animals; D002417:Cattle; D005591:Chemical Fractionation / Q000379:methods*; D012085:Chymosin / Q000302:isolation & purification*; D004926:Escherichia coli / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000201:enzymology; D010710:Phosphates; D011092:Polyethylene Glycols; D011108:Polymers; D011134:Polysaccharides; D011409:Propylene Glycols; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000302:isolation & purification; D012965:Sodium Chloride; D012995:Solubility; D014867:Water
D010710:Phosphates; D011108:Polymers; D011134:Polysaccharides; D011409:Propylene Glycols; D011994:Recombinant Proteins; D014867:Water; C012504:polypropylene glycol; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C008315:maltodextrin; D012085:Chymosin