Partitioning of alkaline protease from Bacillus licheniformis (ATCC 21424) using PEG–K2HPO4 aqueous two-phase system

Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system

“…However, for industrial scale, such procedures are not considered profitable due their high cost and long processing time [11]. Thus, the search for alternative protease purification methods has become increasingly common [12]. Liquid-liquid extraction using aqueous two-phase systems (ATPS) is one of the most promising bioseparation techniques which can, although being simple and inexpensive, be used in place of difficult solid-liquid separation processes, in the initial or subsequent purification steps [13].…”

Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization

“…The pH of the citrate solution was regulated to 6.0 by combining required amounts of citric acid monohydrate and tri-sodium citrate dehydrate, following which, an adequate amount of distilled water was added to the system to get a final mass of 10 g system. The ATPS mixture was stirred carefully and centrifuged (Sartorius Model 3-18k, Sartorius AG, Weender Land Strasse, Gottingen, Germany) at 3000 rpm and 25 °C for 10 min, in order to make thermodynamic equilibrium between two phases [67]. Consequently, the two phases became clear and transparent, and the interface became extremely noticeable.…”

Evaluation of Driving Forces for Protein Partition in PEG-Salt Aqueous Two- Phase Systems and Optimization by Design of Experiments

“…As the PEG chain length is increased, there will be less hydroxyl groups available for the same polymer concentration, thus increasing the hydrophobicity in the top phase [49,58,59]. Hence, owing to an increasing hydrophobicity, proteins (e.g., α-CT/LYZ) with a large hydrophobic surface area are selectively partitioned into the PEG-rich top phase, thus increasing K [6,60,61]. Moreover, a rising PEG concentration is resulting in an increasing number of polymer units involved in protein partition, thus favoring protein (e.g., α-CT/ LYZ) partitioning into the polymer-rich top phase and increasing K due to rising hydrophobic interactions between PEG and protein which prevail over the excluded volume effect [45,62].…”

“…In this context, a pH change is influencing the ionization of the protein side chains containing several ionizable groups, thus changing the surface net charge of the target protein and the hydrophobic-hydrophilic balance of the surface, and finally their K [76,87]. That means, the partitioning of charged proteins in ATPS is affected by their surface net charge which itself is depending on the pH of the solution [12,49,60]. Hence, it is important to consider the nature of charged amino-acid residues at the protein surface to understand the relationship between K and the pH of the system [47,88].…”

“…Moreover, the partitioning of a protein in two aqueous phases is depending on its isoelectric point (pI), i.e., whether pH<pI (positive net charge), pH>pI (negative net charge), or pH=pI (zero net charge) [13,60,67,89]. Consequently, proteins become less positively or more negatively charged by rising pH [12,51].…”