CUTINASE PURIFICATION ON POLY(ETHYLENE GLYCOL) HYDROXYPROPYL STARCH AQUEOUS 2-PHASE SYSTEMS

The partition behaviour of cutinase on poly(ethylene glycol) (PE%)-hyd roxypropyl starch aqueous two-phase systems was characterized. The eff ect 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 s tarch (Reppal PES 100) and a crude one (HPS). The effect of the presen ce of different salts, such as sodium chloride, sodium sulphate and am monium sulphate, on cutinase partition was also studied. The results l ead to the conclusion that aqueous two-phase systems composed of PEG a nd hydroxypropyl starch are not efficient in the purification of cutin ase. In the majority of cases, the partition coefficients were very cl ose to 1, with pH being the factor which affects most cutinase partiti on. 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)(2)SO4], the partition coefficient for cutinase was 3.7, while a v alue of 12 was obtained for PEG 4000-HPS (at pH 4.0; 1 M NaCl). An iso electric point (pl) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts. (C) 1998 Elsevier Science B.V. All rights reser ved.