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Development of a process for large-scale purification of C-phycocyanin from Synechocystis aquatilis using expanded bed adsorption chromatography

Author:
Ramos, Amparo, Acién, F.Gabriel, Fernández-Sevilla, José M., González, Cynthia V., Bermejo, Ruperto
Source:
Journal of chromatography 2011 v.879 no.7-8 pp. 511-519
ISSN:
1570-0232
Subject:
Synechocystis, absorption, adsorbents, adsorption, ion exchange chromatography, osmotic stress, pH, phycocyanin, polyacrylamide gel electrophoresis, purification methods, reversed-phase high performance liquid chromatography, spectroscopy, viscosity
Abstract:
In this paper a large and scaleable method for purification of C-phycocyanin (C-PC) from the cyanobacteria Synechocystis aquatilis has been developed. Phycobiliproteins are extracted from the cells by osmotic shock and separated by passing the centrifuged cell suspension through an expanded bed adsorption chromatography (EBAC) column using Streamline-DEAE as adsorbent. The eluted C-PC rich solution is finally purified by packed-bed chromatography using DEAE-cellulose. Optimal extraction is achieved using phosphate 0.05M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15mm internal diameter (I.D.). The optimal conditions are a sample load of 4.9mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40, 60 and 90mm) and the success of the scale-up process is verified by determining the protein breakthrough capacity and product recovery. The yield of the EBAC step is in the range of 90–93% for every column diameter. To obtain pure C-PC, conventional ion-exchange chromatography with DEAE-cellulose is utilized and a yield of 74% is obtained. The overall yield of the process, comprising all steps, is 69%. The purification steps are monitored using SDS-PAGE and the purity of recovered C-PC is confirmed by absorption and emission spectroscopy and RP-HPLC. Results show that EBAC method is a scalable technology that allows large quantities of C-PC to be obtained without product loss, maintaining a high protein recovery while reducing both processing cost and time.
Agid:
1003420