Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)–sodium citrate aqueous two-phase systems

Abstract

Studies on the partition and purification of penicillin acylase from osmotic shock extract Escherichia coli were performed in poly (ethylene glycol)-citrate systems. Both partition behavior of the enzyme and total protein are similar to those described in other reports increasing ,with pH and tie-line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie-line at pH 6.9. In these conditions 2.6 fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determine the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Althought protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7 fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most of contaminants proteins

Partial purification of ?-amylase from culture supernatant of Bacillus subtilis in aqueous two-phase systems

“…The data show that K a and K t both decrease with increasing PEG molecular weight, which agrees with the observations of Schmid et al [2] and Marcos et al [9]. As the degree of PEG polymerization increases, there are fewer hydroxyl groups for the same concentration of PEG, which leads to an increase in hydrophobicity in the PEG-richer upper phase.…”

“…The phase-forming salt is totally composed of sodium citrate at pH 7.2. The tie line length for the same system composition (PEG and phase-forming salt concentration) decreased as the pH was lowered from 7.4 to 5.2, and the ATPS with a short tie line length did not even form two phases at a low pH of 5.2, which correlates with the idea that trivalent citrate ions are more effective for phase separation [9]. The partition coefficients for α-amylase (K a ) increase with pH for short and long tie line lengths and reach a maximum at around pH 6.6, whereas the partition coefficient remained almost constant for a medium length tie line (Fig.…”

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

“…In fact, as PEG chain length increased there were less hydroxyl groups for the same concentration of the polymer and hence the polymer-richer upper phase increased in hydrophobicy. 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.…”

Purification of C-phycocyanin from Spirulina platensis in aqueous two-phase systems using an experimental design

“…Hence, the aqueous two-phase system (ATPS), a simple and economic system, has been studied. This system is a better alternative for many biomolecules in bioseparation processes, especially in the early downstream stages, and consists of the mixture of two polymers or of one polymer with a salt in a determined concentration, which assures the formation of two phases at equilibrium (Albertson 1986;Marcos et al 1999). The high water content of an aqueous two-phase system (ATPS) provides a neutral and non-toxic environment favorable to biological activities.…”