A two-step approach combining an aqueous two-phase system (ATPS) and an aqueous micellar two-phase system (AMTPS), both based on the thermo-responsive copolymer Pluronic L-35, is here proposed for the purification of proteins and tested on the sequential separation of three model proteins, cytochrome c, ovalbumin and azocasein. Phase diagrams were established for the ATPS, as well as co-existence curves for the AMTPS. Then, by scanning and choosing the most promising systems, the separation of the three model proteins was performed. The aqueous systems based on Pluronic L-35 and potassium phosphate buffer (pH = 6.6) proved to be the most selective platform to separate the proteins (SAzo/Cyt = 1667; SOva/Cyt = 5.33 e SAzo/Ova = 1676). The consecutive fractionation of these proteins as well as their isolation from the aqueous phases was proposed, envisaging the industrial application of this downstream strategy. The environmental impact of this downstream process was studied, considering the carbon footprint as the final output. The main contribution to the total carbon footprint comes from the ultrafiltration (~ 49%) and the acid precipitation (~ 33%) due to the energy consumption in the centrifugation. The ATPS step contributes to ~ 17% while the AMTPS only accounts for 0.30% of the total carbon footprint.

中文翻译：

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水（胶束）两相系统在蛋白质分离中的整合

本文提出了一种基于水敏性共聚物Pluronic L-35的水两相系统（ATPS）和胶束两相系统（AMTPS）的两步法，用于蛋白质的纯化并进行了测试顺序分离三种模型蛋白：细胞色素c，卵清蛋白和偶氮酪蛋白 建立了ATPS的相图以及AMTPS的共存曲线。然后，通过扫描并选择最有希望的系统，进行了三种模型蛋白的分离。事实证明，基于Pluronic L-35和磷酸钾缓冲液（pH = 6.6）的水性体系是分离蛋白质的最具选择性的平台（SAzo / Cyt = 1667； SOva / Cyt = 5.33 e SAzo / Ova = 1676）。提出了这些蛋白质的连续分级分离方法以及从水相中分离的方法，设想此下游策略的工业应用。研究了该下游工艺对环境的影响，并将碳足迹作为最终产出。对总碳足迹的主要贡献来自超滤（〜49％）和由于离心过程中的能量消耗而导致的酸沉淀（〜33％）。ATPS步骤贡献了约17％，而AMTPS仅占总碳足迹的0.30％。