resumo
BACKGROUND: Self-buffered aqueous biphasic systems (ABS) are prepared with ionic liquids (ILs) based on Good's buffers (GB-ILs) and used to purify proteins. RESULTS: A set of new GB-ILs based on the tetrabutylphosphonium cation ([P-4444](+)) was here synthesized and characterized. The self-buffering behaviour of the GB-ILs was asserted by measuring their protonation constants by potentiometry. These ionic liquids display self-buffering characteristics as well as a low toxicity towards the luminescent bacteria Vibrio fischeri. The ability of the GB-ILs to form ABS with the potassium citrate salt was investigated. These systems were then evaluated to extract and purify BSA from bovine serum samples. Extraction efficiencies of 100+/-5% of BSA to the GB-IL-rich phase were obtained in a single step. In addition, good recovery yields (59.4+/-0.8% to 80.1+/-0.7%), and purity levels of BSA (75.0+/-0.3% to 92+/-1%) were also achieved. The BSA secondary structure in the aqueous IL-rich solutions was evaluated through infrared spectroscopic studies revealing the protein-friendly nature of the synthesized GB-ILs. Dynamic light scattering (DLS), 'Conductor-like Screening Model for Real Solvents' (COSMO-RS), and molecular docking studies were carried out to better understand the main driving forces of the extraction process. CONCLUSION: The results obtained show that GB-IL based ABS can be prepared and used for protein purifications, with the GB-ILs showing a stabilizing effect on the protein. (C) 2017 Society of Chemical Industry
palavras-chave
BOVINE SERUM-ALBUMIN; 2-PHASE SYSTEMS; LIPASE PURIFICATION; PROTEIN SEPARATION; GLASS-ELECTRODE; COSMO-RS; EXTRACTION; STABILITY; FLUIDS; SALTS
categoria
Biotechnology & Applied Microbiology; Chemistry; Engineering
autores
Taha, M; Quental, MV; Silva, FAE; Capela, EV; Freire, MG; Ventura, SPM; Coutinho, JAP
nossos autores
Ana Francisca Osório de Almeida Coelho e Silva
Investigador Júnior
Emanuel V. Capela
Investigador Júnior
Mara Guadalupe Freire Martins
Investigador Coordenador
Maria João Quental
Bolseiro de pós-Doutoramento
Mohamed Taha Ahmed Amin
Bolseiro de pós-Doutoramento
Sónia Patrícia Marques Ventura
Professor AuxiliarGrupos
G1 - Materiais Porosos e Nanossistemas
G4 - Materiais Renováveis e Economia Circular
G5 - Materiais Biomiméticos, Biológicos e Vivos
Projectos
CICECO - Aveiro Institute of Materials (UID/CTM/50011/2013)
Projeto de Investigação Exploratória: Sónia Patricia Marques Ventura (IF/00402/2015)
agradecimentos
This work was developed in the scope of the projects CICECO - Aveiro Institute of Materials (Ref. FCT UID/CTM/50011/2013) and PTDC/ATP-EAM/5331/2014, financed by national funds through the FCT/MEC and co-financed by FEDER under the PT2020 Partnership Agreement. Authors are thankful to FCT for financial support through the doctoral and post-doctoral grants of SFRH/BD/94901/2013, and SFRH/BPD/78441/2011 of F.A. e Silva, and M. Taha, respectively. M.G. Freire acknowledges the European Research Council (ERC) for the Grant ERC-2013-StG-337753. S.P.M. Ventura acknowledges FCT for the contract IF/00402/2015 under the Investigador FCT 2015.