CONICET | Buscador de Institutos y Recursos Humanos

To describe the phase separation of micellar solutions containing the non ionic surfactant Triton X-114 and Pichia pastoris supernatant the corresponding coexistence curves were mapped out. The effect of supernatant mass and the presence of different electrolytes and protein binding molecules over the curves characteristics were also investigated. The coexistence curves were determined using the cloud-point method. Specifically, this method consists of visually identifying the temperature (Tcloud) at which solutions of known surfactant concentrations become turbid as the temperature is raised. Experimentally, buffered solutions of a total mass of 3 g, were prepared in graduated 15 mL test tubes by the addition of the desired amounts of triton X-114 and fermentation supernatant. The solutions were well mixed during 1 hour and equilibrated at 4 °C in a thermostated bath in order for each solution to have a clear and homogeneous single phase. Subsequently, the cloud point was obtained by gradually heating up the solution. Solid salts and protein binding molecules solutions were then added. The systems showed temperatures of phase separation lower than 30 °C. Generally, the shapes of the coexistence curves were in good accordance with those published by authors using the cloud point method. The increase of fermentation supernatant mass resulted in a drastic decrease of the cloud temperature but maintaining a similar shape. Similar results were observed to the addition of NaCl, MgSO4 and Cibacrom Blue F3GA. On the other hand, solutions containing KI showed higher Tcloud because of the salting in effect of the iodide anion. Due to the low temperatures of phase separatation, the micellar systems were considered suitable to the extraction of proteins present in P. Pastoris fermentation supernatant by the principle of two phase partitioning.

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