Bromelain partitioning in two-phase aqueous systems containing PEO-PPO-PEO block copolymers.

Bromelain is an enzymatic complex obtained from pineapple (Ananas comosus) fruits and stem. Thermoseparation of bromelain by poly(ethylene oxide) (PEO)- poly(propylene oxide) (PPO)- poly(ethylene oxide) (PEO) block copolymers aqueous solutions was studied. Triblock copolymers with different EO percentages and different molecular mass were evaluated. Copolymer solutions at different pH values, buffer concentrations and copolymer concentrations were investigated. It was found that cloud point temperature increases as a function of %EO and decreases with copolymer molecular mass, copolymer concentration and buffer concentration. The results showed that all the studied factors influenced enzyme partition. The best conditions were copolymer with 10% EO and molecular mass of 2000 g/mol, temperature of 25 degrees C, copolymer concentration of 5% (w/w), pH 6.0 and salt concentration of 15 mM. Enzyme activity recovery around 79.5%, purification factor around 1.25 and activity partition coefficient around 1.4 were obtained.

[1]  H. Tani,et al.  Phase separation in aqueous micellar solutions of nonionic surfactants for protein separation , 1995 .

[2]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[3]  F. Tjerneld,et al.  Application of temperature-induced phase partitioning at ambient temperature for enzyme purification. , 1994, Journal of chromatography. A.

[4]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[5]  A. Hernández-Arana,et al.  The thermal denaturation of stem bromelain is consistent with an irreversible two-state model. , 1995, Biochimica et biophysica acta.

[6]  G. Karlstroem.  A new model for upper and lower critical solution temperatures in poly(ethylene oxide) solutions , 1985 .

[7]  B. Mattiasson,et al.  Aqueous two-phase system formed by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail , 1997 .

[8]  Pressure- and temperature-dependent micellization of a poly(ethylene oxide)poly(propylene oxide) triblock copolymer in aqueous solutions , 1996 .

[9]  K. Berggren,et al.  Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers , 1995 .

[10]  J. Persson,et al.  Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers. , 1999, Journal of chromatography. A.

[11]  Folke Tjerneld,et al.  Purification of recombinant proteins using thermoseparating aqueous two‐phase system and polymer recycling † , 1999 .