Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum

Abstract

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe and analytical reagent. In this paper, B-phycoerythrin and R-phycocyanin in native state, from the red alga Porphyridium cruentum were obtained by an inexpensive and simple process. The best results of this purification procedure were scaled up by a factor of 13 to a large preparative level using an anionic chromatographic column of DEAE cellulose. Gradient elution with acetic acid-sodium acetate buffer (pH 5.5) was used. In these conditions both 32% of B-phycoerythrin and 12% of R-phycocyanin contained in the biomass of the microalgae was recovered. B-phycoerythrin was homogeneous as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), yielding three migrating bands corresponding to its three subunits, consistent with the (αβ)₆γ subunit composition characteristic of this biliprotein and the spectroscopic characterization of B-PE (UV–visible absorption and emission spectroscopy; steady-state and polarization fluorescence), is accompanied. Finally, a preliminary cost analysis of the recovery process is presented.

Introduction

The phycobiliproteins are proteins with linear tetrapyrrole prosthetic groups (bilins) that, in their functional state, are covalently linked to specific cysteine residues of the proteins. These proteins are found in cyanobacteria (blue-green algae), in a class of biflagellate unicellular eukaryotic algae (cryptomonads), and in Rhodophyta (red algae). In all of them the phycobiliproteins act as photosynthetic accessory pigments. Phycobiliproteins are used as colorants in food (chewing gums, dairy products, ice sherbaths, gellies, etc.) and cosmetics such as lipstick and eyeliners in Japan, Thailand and China (Dainippon Ink and Chemicals, 1985). It was also shown to have therapeutic value by immunomodulating activity and anticancer activity (Iijima and Shimamatsu, 1982). Owing to its fluorescence properties it has gained importance in the development of phycofluor probes for immunodiagnostics (Kronick and Grossman, 1983). In this sense, the red marine microalga Porphyridium cruentum is a Rhodophyta of increasing interest as a source of valuable phycobiliproteins, as well as sulphated exopolysaccharides, superoxide-dismutase, and polyunsaturated fatty acids (Vonshak, 1988) with applications in the food and pharmaceutical industries.

The phycobiliproteins are assembled into an organized cellular structure, the phycobilisome. They absorb light, over a wide range of wavelengths in the visible part of the spectrum, and transfer the excitation energy by radiationless processes to the reaction centres in the photosynthetic membranes for conversion to chemical energy (Gantt, 1980, Glazer, 1976, Glazer and Wedemayer, 1995). The phycobiliproteins can be divided into three main classes depending on absorption properties: phycoerythrins (PE, λ_max 540–570 nm), phycocyanins (PC, λ_max 610–620 nm), and allophycocyanins (APC, λ_max 650–655 nm). Some cyanobacteria have a fourth type of biliprotein in place of PE, the phycoerythrocyanin (Gantt, 1981, Glazer, 1981). Visually, the phycoerythrins appear red, the phycocyanins range from purple (phycoerythrocyanin, R-phycocyanin) to deep blue (C-phycocyanin), and the allophycocyanins are blue with a hint of green. Phycoerythrins can be divided into three main classes, depending on their absorption spectrum, B-phycoerythrin (peaks at 545, 565 nm with a 499 shoulder), R-phycoerythrin (peaks at 499, 565 nm and a 545 shoulder) and C-phycoerythrin (peak at 565) (Glazer, 1984, Hilditch et al., 1991, Galland-Irmouli et al., 2000).

The introduction of phycobiliproteins as fluorescent tags of cells and macromolecules was followed by its widespread application in highly sensitive fluorescence techniques such as fluorescent activated cell sorting, flow cytometry, fluorescence immunoassay and fluorescence microscopy (Glazer and Stryer, 1984). B-PE has been shown to be particularly useful due to its large absorption coefficient and great fluorescence properties just as the high quantum yield and high Stokes shift. B-PE fluoresces in a spectral region that is distinct from the region of emission of the simple organic dyes commonly used as fluorescent indicators. Therefore, B-PE is a valuable candidate in the design and characterization of light-sensing elements in biosensors (Ayyagari et al., 1995). Another interesting application of the phycobiliproteins is their use as natural dyes in foods and cosmetics replacing the synthetic dyes, since these are in general toxic, carcinogenic or otherwise unsafe. In Japan where algal cultivation is a well-developed industry, some natural pigments from phycobiliproteins have already been patented (Dainippon Ink and Chemicals, 1979, Dainippon Ink and Chemicals, 1987). Other colorants prepared from algae are also suitable for use in cosmetics (Arad and Yaron, 1992). Again, B-PE is the most valuable of the phycobiliproteins due to its intense and unique pink colour. On the other hand, its utilization as natural dyes in some kinds of heat-treated foods or in micelle solubilization makes it convenient to obtain smaller aggregates than hexameric B-PE (Bermejo et al., 2000).

Pure phycobiliproteins from crude algal extracts are usually obtained by a combination of different and non-scaleable methods (Gray and Gantt, 1975, Grabowski and Gantt, 1978, Duerring et al., 1991, Ficner et al., 1992, Bermejo et al., 1997). Particularly, phycoerythrin is classically purified by a combination of several techniques such as ammonium sulphate precipitation, ion-exchange chromatography, gel filtration and chromatography on hydroxylapatite (Hilditch et al., 1991, D'Agnolo et al., 1994, Schoelember et al., 1983, Ficner et al., 1992). Purification procedures are often long and complex. For this reason the use of this biliprotein has been somewhat limited by the tedious preparation of adequate amounts of the purified protein.

In view of the multiple uses of phycoerythrin, in the present work a scaleable methodology for purification of large amounts of pure β-phycoerythrin from the red algae P. cruentum is proposed. The yield of the process at various stages is analysed. The high purity of B-PE obtained was confirmed by SDS-PAGE electrophoresis and the spectroscopic characterization of B-PE. Finally, the economic aspects of the process are discussed.