Selective Separation of Proteins Using Stimuli-Responsive Polymers in an Aqueous System
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Biopolymers provide an option for protein purification and are being used due to their biodegradability, cost efficiency, non-toxicity and abundance in nature. This study investigated the use of natural and synthetic polymers for the flocculation of proteins by evaluating two bioprocesses: polyelectrolyte precipitation and inverse transition cycling. The effectiveness of chitosan (CHI, pKa= 6.5), polyetheleneimine (PEI, pKa= 11), sodium alginate (ALG, pKa= 3.2), and poly-N-isopropylacrylamide (PNIPAAm, lower critical solution temperature= 32 °C) were evaluated as flocculating agents of two model proteins, bovine serum albumin (BSA, pI=4.5, acidic protein) and lysozyme (LYZ, pI= 11, basic protein). Natural polymers, CHI and ALG have an advantage due to their stimuli-responsive characteristic and also because they are positively or negatively charged at certain pH values, while PNIPAAm, a synthetic polymer, is sensitive to temperature. Briefly, protein and polymer were mixed at pH values where charges (if applicable) were complementary, ideally triggering flocculation by electrostatic interactions. The maximum precipitation yield achieved by polyelectrolyte precipitation resulted from the flocculation of LYZ using ALG at 87%. Our model system included the use of PEI as a flocculant for BSA. This resulted in a 65% precipitation yield. In comparison, CHI was evaluated as a natural flocculant for BSA, which resulted in a 61% precipitation yield. If flocculation did not occur, a phase transition change of polymer was induced by triggering the appropriate stimulus, i.e., temperature, pH, or ionic strength, to precipitate proteins by sweep flocculation. We attempted to use PNIPAAm to precipitate both BSA and LYZ; however, precipitation yield was less than 10%. A subsequent study addressed the synthesis, characterization, and use of PNIPAAm based copolymers and blends for protein flocculation, and it was concluded that these copolymers and blends were not as effective as the single polymers (less than 10% increase in precipitation yield). Lastly, optimized bioprocess for precipitation using PEI resulted in a precipitation yield of 37% of all milk proteins. Overall, this study showed that natural polymers (CHI and ALG) can produce precipitation yields comparable to the standard method provided by PEI and should be considered as an alternative for flocculation of proteins.
Johnson, Jecori Shana (2018). Selective Separation of Proteins Using Stimuli-Responsive Polymers in an Aqueous System. Master's thesis, Texas A & M University. Available electronically from