Preparation of high-purity R-phycoerythrin and R-phycocyanin from Pyropia yezoensis in membrane chromatography

Abstract

R-phycoerythrin and R-phycocyanin are the main light-harvesting pigment-protein complexes in Pyropia yezoensis. They have bright colors and a high degree of fluorescence, and are now widely used in biomedicine, food, and cosmetic industries. Therefore, membrane chromatography was used to isolate and purify phycobiliprotein from P. yezoensis. Algal cells were disrupted by repeated freeze-thaw cycles. Then ammonium sulfate was added into the crude phycobiliprotein extract, so that the saturation of ammonium sulfate in the solution reached 20%, 30%, and 40% in sequence. At 30% and 40% ammonium sulfate saturation, precipitates of R-phycoerythrin and R-phycocyanin mixture in different proportions were obtained. The precipitates were ultra-filtrated to remove the salt, and then, the retentate was taken for membrane chromatographic treatment. In the membrane chromatographic treatment, salt-sensitive PVDF (polyvinyl difluoride) membranes were used for the follow-up experiments. By adjusting the concentration of ammonium sulfate, the membrane system changed the binding strength of various proteins, and then separated different types of phycobiliproteins. The final products of R-phycoerythrin (Aλmax/A280 purity 4.25, yield 45%) and R-phycocyanin (two purity grades: purity 5.8, yield 14.70% and purity 4.18, yield 39.89%) were obtained and then characterized by spectrophotometric and fluorometric measurements and SDS-PAGE. However, the large-scale production of R-phycocyanin and R-phycoerythrin remains a bottleneck as the column chromatography is difficult to be applied for industrial production. This method avoids the problem of easy clogging of column chromatography and can be useful for mass-production of R-phycoerythrin and R-phycocyanin in the future based on the laboratory results.