Cutinase purification on poly(ethylene glycol)-hydroxypropyl starch aqueous two-phase systems.
The partition behaviour of cutinase on poly(ethylene glycol) (PEG)-hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000-Reppal PES 100 [at pH 4.0; 0.5 M (NH4)2SO4], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000-HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.
Departamento de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, Braga Codex, Portugal.
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Almeida
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Venâncio
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Teixeira
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Aires-Barros
D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D019855:Ethylene Glycol; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013213:Starch / Q000031:analogs & derivatives; D014867:Water
C103009:Reppal PES 100; D014867:Water; D013213:Starch; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D019855:Ethylene Glycol