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http://hdl.handle.net/10773/35617
Title: | In situpurification of periplasmatic L-asparaginase by aqueous two phase systems with ionic liquids (ILs) as adjuvants |
Author: | Santos, João HPM Flores-Santos, Juan C Meneguetti, Giovanna P Rangel-Yagui, Carlota O Coutinho, João AP Vitolo, Michele Ventura, Sónia PM Pessoa, Adalberto |
Keywords: | Therapeutic enzyme L-asparaginase Aqueous two-phase systems Ionic liquids as adjuvants Integrated in situ purification process |
Issue Date: | 2018 |
Publisher: | Wiley |
Abstract: | (ALL) and lymphosarcoma. Considering its main use in cancer therapy, the most important request for ASNase production is the need for a highly pure biopharmaceutical obtained in the final of the downstream process, which is considered as the crucial step in its production. RESULTS: This work proposes the use of polymer–salt aqueous two-phase systems (ATPS) based on polyethylene glycol and citrate buffer, with ionic liquids (ILs) as adjuvants, combined with the permeabilization of cell membrane using n-dodecane and glycine for the in situ purification of periplasmatic ASNase from Escherichia coli cells. The process proposed was optimized (polymer molecular weight, pH, tie-line length/mixture point, presence, nature and concentration of the adjuvant). The results show that ASNase partitions mostly to the PEG-rich phase, due to hydrophobic interactions between both PEG and enzyme. Remarkably, the addition of 5 wt% of 1-butyl-3-methylimidazolium methanesulfonate [C4mim][CH3SO3] as adjuvant lead to high recoveries [87.94 ± 0.03 (%)], purification factors (20.09 ± 0.35), and a final specific activity SA = 3.61 ± 0.38 U mg-1 protein, from a crude enzyme extract with a SA = 0.18 ± 0.05 U mg-1 protein. Moreover, better results were achieved when a prepurification step consisting of an ammonium sulfate precipitation was combined with the optimized ATPS, achieving an increased SA = 22.01 ± 1.36 U mg-1 protein and PF = 173.8. CONCLUSIONS: A novel integrated downstream process was successfully implemented for the in situ purification of ASNase from fermentation broth. |
Peer review: | yes |
URI: | http://hdl.handle.net/10773/35617 |
DOI: | 10.1002/jctb.5455 |
ISSN: | 0268-2575 |
Appears in Collections: | CICECO - Artigos |
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C.pdf | 2.68 MB | Adobe PDF |
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