Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

Abstract

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 (NH) SO ], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000-HPS (at 4 2 4 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.

Extraction of amylase from fermentation broth in poly (Ethylene Glycol) salt aqueous two-phase system

“…On the other hand, the chain length increase also caused the increase of the excluded volume, which mean less space available for the protein in the PEG rich phase (Marcos et al 1999). Similar behavior was observed by Marcos et al (1999) and Almeida et al (1998) studing the purification of penicillin acylase from Escherichia coli and cutinase from E. coli WK-6 recombinant, respectively, using poly (ethylene glycol)-sodium citrate and poly (ethylene glycol)-hydroxypropyl starch.…”

“…The same didn't occur in PEG 8000 system at pH 8.0, where there was no significant difference in the K value. Study carried by Almeida et al (1998), Furuya et al (1995 and Chaves et al (2002) for the extraction of cutinase in the PEG 4000/hydroxypropyl starch, hydrolytic enzyme in PEG-dextran system and extraction of Sm-13 recombinant antigen from Escherichia coli in the PEG (1000, 3350 and 8000)/Reppal PES 100 system, respectively, showed that the partition coefficients were significantly influenced by the PEG molecular weight. Ortin et al (1991), studying the partition of α-lactoalbumin and β-lactoglobulin in PEG/hydroxypropyl starch systems, showed that the increase of the tie line resulted protein displacement to the bottom phase, thus decreasing the K value.…”

Direct purification of Burkholderia Pseudomallei lipase from fermentation broth using aqueous two-phase systems

“…It appeared that the low partition efficiency of ATPS with PEG 8000 could be caused by the effect of excluded volume [23]. Precipitation at the interphase was observed since the protein saturation point in the polymer phase had reached.…”

Practical application of aqueous two-phase partition to process development for the recovery of biological products

“…Here again, the system pH that results in an increase in product recovery must be considered. The general practical strategies outline here has been exploited for the development of recent ATPS processes (as examples see references [7,11,16,20]). It is important to consider that, if after the manipulation of the different system parameters the product recovery achieved is not acceptable, then a change of the conditions of the selected ATPS (e.g.…”