Abstract
Aqueous Two Phase System (ATPS) or liquid-liquid extraction is used in biotechnology to recover valuable compounds from raw sources. In Aqueous Two-Phase Systems, many factors influence the Partition coefficient, K, (which is the ratio of protein concentration in the top phase to that in the bottom phase) and the Recovery percentage (Rec%). In this research, two systems of ATPS were used: first, polyethylene glycol (PEG) 4000/Sodium citrate (SC), and the second, PEG8000/ Sodium phosphate (SPH), for the extraction of Bovine Serum Albumin (BSA). The behavior of Rec% and K of pure (BSA) in ATPS has been investigated throughout the study by the effects of five parameters: temperature, concentration of polyethylene glycol (PEG4000 and PEG8000), the concentration of Sodium citrate or Sodium phosphate, pH, and the addition of sodium chloride as a supporting agent. The recovery percentage of BSA and its partition coefficient are significantly influenced by these factors to various degrees. The most influential variable in this study is PEG concentration for both systems. In addition to the PEG concentration, the stabilizing impact of NaCl is a crucial factor. The interaction between biomolecules and PEG gets more hydrophobic as the PEG concentration is raised. In the first system (PEG4000/SC), the maximum recovery percentage and partition coefficient were 98.99% and 97.69, respectively, at 31°C, PEG4000 concentration 1.5g/10 ml, Sodium citrate concentration 2.7 g/10 ml, pH 10, and 0.5 M NaCl concentration. While in the second system (PEG8000/SPH), the maximum recovery percentage and partition coefficient was 98.93% and 92.12, respectively, at 31oC, PEG8000 concentration 1.5 g/10 ml, Sodium phosphate concentration 2.4 g/10 ml, pH 10, and concentration of NaCl 0.5 M.
References
P.-Å. Albertsson, Partition of cells particles and macromolecules in polymer two-phase systems, 2nd editio., vol. 24. New York: Wiley- Interscience, 1970.
A. L. Grilo, M. R. Aires-Barros, A. M. Azevedo, A. Glyk, T. Scheper, and S. Beutel, “Partitioning in Aqueous Two-Phase Systems: Fundamentals, Applications and Trends,” Sep. Purif. Rev., vol. 45, no. 1, pp. 68–80, 2016, doi: 10.1080/15422119.2014.983128.
R. Hatti-kaul, Methods in biotechnology Aqueous Two-Phase Systems - Methods and Protocols, vol. 11. 2000. [Online]. Available: http://linkinghub.elsevier.com/retrieve/pii/B9780080885049001240
P.- Albertsson et al., Partitioning in Aqueous Two-Phase System. 1985. doi: 10.1016/b978-0-12-733860-6.x5001-4.
J. A. Asenjo and B. A. Andrews, “Aqueous two-phase systems for protein separation: A perspective,” J. Chromatogr. A, vol. 1218, no. 49, pp. 8826–8835, 2011, doi: 10.1016/j.chroma.2011.06.051.
R. Hatti-kaul, “Aqueous Two-Phase Systems,” vol. 19, 2001.
E. Espitia-saloma, P. Vázquez-villegas, O. Aguilar, and M. Rito-palomares, “Continuous aqueous two-phase systems devices for the recovery of biological products,” Food Bioprod. Process., 2013, doi: 10.1016/j.fbp.2013.05.006.
J. V. D. Molino, D. de A. V. Marques, P. G. Mazzola, and Maria Silvia Viccari Gatti, “DIFFERENT TYPES OF TWO-PHASE SYSTEMS FOR BIOMOLECULE AND BIOPARTICLE EXTRACTION AND PURIFICATION João Vitor Dutra Molino Daniela de Araújo Viana Marques Priscila Gava Mazzola Maria Silvia Viccari Gatti Adalberto Pessoa Júnior Upstream improvements have le,” 2013, doi: 10.1002/btpr.
M. SG, “The Influence of Different Molecular Weights of Polyethylene Glycol ( Peg ) to the Formation of the Aqueous Two-Phase System Peg-Natrium Salt of Citric Acid-Water,” COJ Biomed. Sci. Res., vol. 1, no. 3, pp. 1–3, 2020, doi: 10.31031/COJBSR.2020.01.000512.
S. H. Ammar, W. A. Abdul-nabi, and M. K. Rasheed, “Extraction of Zn ( II ) and Cu ( II ) Ions Using PEG ( 300 ) - KCl Salt Aqueous Two-Phase Systems,” vol. 7, no. 2, pp. 68–74, 2011.
A. G. Teixeira, R. Agarwal, K. R. Ko, J. Grant-burt, B. M. Leung, and J. P. Frampton, “Emerging Biotechnology Applications of Aqueous Two-Phase Systems,” vol. 1701036, pp. 1–19, 2017, doi: 10.1002/adhm.201701036.
Y. K. Yau, C. W. Ooi, E. P. Ng, J. C. W. Lan, T. C. Ling, and P. L. Show, “Current applications of different type of aqueous two-phase systems,” Bioresour. Bioprocess., vol. 2, no. 1, 2015, doi: 10.1186/s40643-015-0078-0.
J. F. B. Pereira, M. G. Freire, and J. A. P. Coutinho, “Aqueous two-phase systems: Towards novel and more disruptive applications,” Fluid Phase Equilib., vol. 505, Feb. 2020, doi: 10.1016/j.fluid.2019.112341.
O. S. Nnyigide and K. and Hyun, “Rheo-kinetics of bovine serum albumin in catanionic surfactant systems.,” Korean J. Chem. Eng., vol. 35, pp. 1969–1978, 2018.
A. Kilara and and V. R. Harwalkar, “Denaturation,” in Food Proteins: Properties and Characterization, S. N. and H. W. M. (Eds.), Ed. New York: VCH Publishers Inc., 1996, pp. 71-165.
O. Nnyigide, “Dissertation for the degree of Doctor of Philosophy A Study of Bovine Serum Dissertation for the degree of Doctor of Philosophy A Study of Bovine Serum Albumin ( BSA ) Hydrogel with Various Chemical Denaturants by Rheological Measurements and Molecular Dy,” 2019.
P. Walstra and R. Jenness, Dairy chemistry & physics. John Wiley & Sons., 1984.
M. Murata, F. Tani, T. Higasa, N. Kitabatake, and and E. Doi, “Heat-induced transparent gel formation of bovine serum albumin.,” Biosci. Biotechnol. Biochem., vol. 57, no. 1, pp. 43–6, 1993.
P. Relkin., “Thermal unfolding of β-lactoglobulin, α-lactalbumin, and bovine serum albumin, A thermodynamic approach,” Crit. Rev. Food Sci. Nutr., vol. 36, no. 6, pp. 565–601, 1996.
P. F. Fox, “Milk protein: General and historical aspects,” in Advanced Dairy Chemistry, Third ed., P. F. Fox and P. L. H. McSweeney, Eds. Boston: Springer, 2003, pp. 1-48. doi: 10.1007/978-1-4419-8602-3_1.
F. Dismer, S. Alexander Oelmeier, and J. Hubbuch, “Molecular dynamics simulations of aqueous two-phase systems: Understanding phase formation and protein partitioning,” Chem. Eng. Sci., vol. 96, pp. 142–151, 2013, doi: 10.1016/j.ces.2013.03.020.
Z. Du, Y. L. Yu, and J. H. Wang, “Extraction of proteins from biological fluids by use of an ionic liquid/aqueous two-phase system,” Chem. - A Eur. J., vol. 13, no. 7, pp. 2130–2137, 2007, doi: 10.1002/chem.200601234.
E. Chandler, “Multi-Stage Aqueous Two-Phase Extraction,” University of Sheffield, 2021.
N. Patel, “Development of aqueous two - phase separations by combining high - throughput screening and process modelling,” no. July, p. 229, 2017.
G. Khayati, “AQUEOUS TWO-PHASE SYSTEMS COMPOSED OF DIFFERENT MOLECULAR WEIGHT PHOSPHATE,” no. January 2012, 2019.
S. Raja and V. R. Murty, “Optimization of Aqueous Two-Phase Systems for the Recovery of Soluble Proteins from Tannery Wastewater Using Response Surface Methodology,” J. Eng. (United Kingdom), vol. 2013, 2013, doi: 10.1155/2013/217483.
Z. M. Jaffer, K. W. Hameed, and S. G. Imran, “Extraction of prodigiosin using aqueous two phase system,” IOP Conf. Ser. Mater. Sci. Eng., vol. 1076, no. 1, p. 012026, 2021, doi: 10.1088/1757-899x/1076/1/012026.
S. Settu, P. Velmurugan, R. R. Jonnalagadda, and B. U. Nair, “Extraction of bovine serum albumin using aqueous two-phase poly (ethylene glycol)-poly (acrylic acid) system,” 2015.
M. Pirdashti, Z. Heidari, N. A. Fashami, S. M. Arzideh, and I. Khoiroh, “Phase equilibria of aqueous two-phase systems of peg with sulfate salt: Effects of ph, temperature, type of cation, and polymer molecular weight,” J. Chem. Eng. Data, vol. 66, no. 3, pp. 1425–1434, Mar. 2021, doi: 10.1021/acs.jced.0c01029.
D. Baskaran, K. Chinnappan, R. Manivasagan, and D. K. Mahadevan, “Partitioning of crude protein from aqua waste using PEG 600-inorganic salt Aqueous Two-Phase Systems,” Chem. Data Collect., vol. 15–16, pp. 143–152, 2018, doi: 10.1016/j.cdc.2018.05.004.
P. A. L. Wijethunga and H. Moon, “On-chip aqueous two-phase system ( ATPS ) formation , consequential self- mixing , and their influence on drop-to-drop aqueous two-phase extraction kinetics,” J. Micromechanics Microengineering, no. March 2016, p. 94002, 2015, doi: 10.1088/0960-1317/25/9/094002.
D. Baskaran, K. Chinnappan, R. Manivasagan, and R. Selvaraj, “Liquid-Liquid Equilibrium of Polymer-Inorganic Salt Aqueous Two-Phase Systems: Experimental Determination and Correlation,” J. Chem. Eng. Data, vol. 62, no. 2, pp. 738–743, Feb. 2017, doi: 10.1021/acs.jced.6b00805.
Y. M. Lu, Y. Z. Yang, X. D. Zhao, and C. B. Xia, “Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systems,” Food Bioprod. Process., vol. 88, no. 1, pp. 40–46, Mar. 2010, doi: 10.1016/j.fbp.2009.12.002.
F. Dumas, E. Roger, J. Rodriguez, L. Benyahia, and J. P. Benoit, “Aqueous Two-Phase Systems: simple one-step process formulation and phase diagram for characterisation,” Colloid Polym. Sci., vol. 298, no. 12, pp. 1629–1636, Dec. 2020, doi: 10.1007/s00396-020-04748-8.
Z. A. S. A.-T. Essam Fadel Alwan Al-Jumaily, Muntaha A. Al-Safar, “Purification and Characterization β - lactamase produce from local isolate Klebsiella pneumonia,” no. March, 2009, [Online]. Available: https://www.researchgate.net/publication/299624173_Purification_and_Characterization_b_lactamase_produce_from_local_isolate_Klebsiella_pneumonia
C. L. Kielkopf, W. Bauer, and I. L. Urbatsch, “Bradford assay for determining protein concentration,” Cold Spring Harb. Protoc., vol. 2020, no. 4, pp. 136–138, Apr. 2020, doi: 10.1101/pdb.prot102269.
M. M. Bradford, “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” 1976.
C. K. Haweel, A. J. Ali, and S. N. Alias, “Separation of Bovine Serum Albumin Using Chromatographical Column : Parameters and Simulation,” vol. 11, no. 1, pp. 1–9, 2010.
S. Saravanan, J. R. Rao, T. Murugesan, B. U. Nair, and T. Ramasami, “Partition of tannery wastewater proteins in aqueous two-phase poly (ethylene glycol)-magnesium sulfate systems: Effects of molecular weights and pH,” Chem. Eng. Sci., vol. 62, no. 4, pp. 969–978, Feb. 2007, doi: 10.1016/j.ces.2006.10.025.
J. He, J. Wan, T. Yang, X. Cao, and L. Yang, “Recyclable aqueous two-phase system based on two pH-responsive copolymers and its application to porcine circovirus type 2 Cap protein purification,” J. Chromatogr. A, vol. 1555, pp. 113–123, 2018, doi: 10.1016/j.chroma.2018.04.032.
V. Ramesh and V. R. Murty, “Partitioning of thermostable glucoamylase in polyethyleneglycol/salt aqueous two-phase system,” Bioresour. Bioprocess., vol. 2, no. 1, pp. 7–11, 2015, doi: 10.1186/s40643-015-0056-6.
M. Iqbal et al., “Aqueous two-phase system (ATPS): an overview and advances in its applications,” Biol. Proced. Online, vol. 18, no. 1, pp. 1–18, 2016, doi: 10.1186/s12575-016-0048-8.
L. Ferreira et al., “Structural features important for differences in protein partitioning in aqueous dextran-polyethylene glycol two-phase systems of different ionic compositions,” Biochim. Biophys. Acta - Proteins Proteomics, vol. 1844, no. 3, pp. 694–704, Mar. 2014, doi: 10.1016/j.bbapap.2014.01.016.
S. Raja, V. R. Murty, V. Thivaharan, V. Rajasekar, and V. Ramesh, “Aqueous Two Phase Systems for the Recovery of Biomolecules – A Review,” Sci. Technol., vol. 1, no. 1, pp. 7–16, 2012, doi: 10.5923/j.scit.20110101.02.
Z. Shad, A. S. M. Hussin, and B. Akbari-adergani, “a Primary Approach for Separation and Characterization of Α-Amylase From White Pitaya (Hylocereus Undatus) Peels By Polymer/Salt Two Phase System,” J. Microbiol. Biotechnol. Food Sci., vol. 11, no. 4, pp. 1–6, 2022, doi: 10.55251/jmbfs.3467.
Z. Shad, H. Mirhosseini, A. S. M. Hussin, B. Forghani, M. Motshakeri, and M. Y. A. Manap, “Aqueous two-phase purification of α-Amylase from white pitaya (Hylocereus undatus) peel in polyethylene glycol /citrate system: Optimization by response surface methodology,” Biocatal. Agric. Biotechnol., vol. 14, pp. 305–313, 2018, doi: 10.1016/j.bcab.2018.01.014.
R. D. Navapara, D. N. Avhad, and V. K. Rathod, “Separation Science and Technology Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two- Phase System Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two-Phase S,” Sep. Sci. Technol., no. August 2014, pp. 37–41, 2011, doi: 10.1080/01496395.2011.578101.
R. S. Faravash, H. Modarress, and B. Nasernejad, “Structural and partitioning studies of bovine serum albumin in mixture of (poly(ethylene glycol) + K2HPO4 + H2O),” J. Chem. Eng. Data, vol. 52, no. 1, pp. 71–76, 2007, doi: 10.1021/je060235b.
F. S. Antelo, J. A. V. Costa, and S. J. Kalil, “Purification of C-phycocyanin from Spirulina platensis in aqueous two-phase systems using an experimental design,” Brazilian Arch. Biol. Technol., vol. 58, no. 1, pp. 1–11, 2015, doi: 10.1590/S1516-8913201502621.
G. Johansson, M. Joelsson, B. Olde, and V. P. Shanbhag, “Affinity partitioning of biopolymers and membranes in ficoll-dextran aqueous two-phase systems,” J. Chromatogr. A, vol. 331, no. C, pp. 11–21, 1985, doi: 10.1016/0021-9673(85)80002-9.
P. E. Kee, J. C. W. Lan, H. S. Yim, Y. H. Chow, P. T. Chen, and H. S. Ng, “Efficiency of polymer/salt aqueous two-phase electrophoresis system for recovery of extracellular Kytococcus sedentarius TWHKC01 keratinase,” Process Biochem., vol. 100, no. September 2020, pp. 199–206, 2021, doi: 10.1016/j.procbio.2020.10.005.
G. Lagomarsino, B. A. Andrews, and J. A. Asenjo, “Effect of electrostatic energy on partitioning of proteins in aqueous two-phase systems,” vol. 807, pp. 81–86, 2004, doi: 10.1016/j.jchromb.2004.03.033.
B. A. Andrews, A. S. Schmidt, and J. A. Asenjo, “Correlation for the Partition Behavior of Proteins in Aqueous Two-Phase Systems : Effect of Surface Hydrophobicity and Charge,” 2005, doi: 10.1002/bit.20495.
G. Johansson, “Aqueous two-phase systems in protein purification,” J. Biotechnol., vol. 3, no. 1–2, pp. 11–18, 1985, doi: 10.1016/0168-1656(85)90003-3.
R. Nuno, L. A. Ferreira, J. A. Teixeira, V. N. Uversky, and B. Y. Zaslavsky, “Effects of sodium chloride and sodium perchlorate on properties and partition behavior of solutes in aqueous dextran-polyethylene glycol and polyethylene glycol-sodium sulfate two-phase systems,” J. Chromatogr. A, pp. 12–14, 2018, doi: 10.1016/j.chroma.2018.11.015.
U. Gündüz, “Partitioning of bovine serum albumin in an aqueous two-phase system: Optimization of partition coefficient,” J. Chromatogr. B Biomed. Sci. Appl., vol. 743, no. 1–2, pp. 259–262, 2000, doi: 10.1016/S0378-4347(00)00068-2.
M. Perumalsamy and T. Murugesan, “Partition behavior of bovine serum albumin in peg2000-sodium citrate-water based aqueous two-phase system,” Sep. Sci. Technol., vol. 42, no. 9, pp. 2049–2065, 2007, doi: 10.1080/15363830701313404.
I. Regupathi, S. Kalaivani, and N. Sindhu, “Bovine Serum Albumin Partitioning in Aqueous Two-Phase Systems: Effects of Variables and Optimization,” Bioprocess. J., vol. 12, no. 1, pp. 29–41, 2013, doi: 10.12665/j121.regupathi.
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