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

作者：Ramos Amparo; Acien F Gabriel; Fernandez Sevilla Jose M; Gonzalez Cynthia V; Bermejo Ruperto^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2011, 879(7-8): 511-519.

DOI：10.1016/j.jchromb.2011.01.013

• Expanded bed adsorption chromatography
• Synechocystis aquatilis
• Phycobiliproteins
• C-phycocyanin
• Preparative protein purification
• Anion-exchange chromatography

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 0.05 M buffer pH 7.0 twice. The operation of EBAC is optimized on a small scale using a column of 15 mm internal diameter (ID.). The optimal conditions are a sample load of 4.9 mg C-PC/mL adsorbent, an expanded bed volume twice the settled bed volume and a sample viscosity of 1.020 mP. The EBAC process is then scaled up by increasing the column I.D. (15, 25, 40,60 and 90 mm) 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 SOS-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.

• 出版日期2011-3-1