Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum
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, λmax 540–570 nm), phycocyanins (PC, λmax 610–620 nm), and allophycocyanins (APC, λmax 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.
Section snippets
Chemicals
Preswollen microgranular DEAE–cellulose, molecular mass standards, dialysis tubing (avg. flat. width 43 mm), dialysis tubing closures, ammonium sulphate and sodium azide were from Sigma diagnostics (St. Louis, MO, USA). The materials used for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were from Pharmacia (Uppsala, Sweden). All other chemicals were from Sigma and used without further purification.
Microalgal biomass
The red microalga P. cruentum UTEX 161 was used. The biomass was obtained
Results
The first phycobiliproteins determination was carried out by precipitation of crude biomass extract with 1% (w/v) streptomycin sulphate for 30 min at 4 °C and centrifugation at 2500×g for 10 min, in order to eliminate membrane fragments containing chlorophyll. The absorption spectrum of this crude extract is shown in Fig. 2. In this spectrum three peaks corresponded to the maxima of B-PE (565, 545 nm and 498 shoulder), the other peak corresponded to the maxima of R-PC (620 nm), and no peak was
Discussion
The phycoerythrin productivity obtained from P. cruentum outdoor cultures was slightly lower than maximum values referenced indoor. Thus, in this work a productivity of 20 mg B-PE per l per day was obtained, much higher than 3 mg per l per day obtained from Calothrix spp. (Olvera-Ramı́rez et al., 2000), but similar to 25 mg per l per day referenced for P. cruentum (Lee and Tan, 1988), or 18 mg per l per day referenced for Gracilaria tenuistipatata (Carnicas et al., 1999), all of them at indoor
Conclusions
With these results we can conclude that the above described purification method is a one step scaleable chromatographic method that provides B-PE solutions in hexameric aggregation state, as well as pure fractions of R-PC. The feasibility of the method has been verified in a 13 fold scale up system, being the only method developed for phycoerythrin. In addition, the methodology described here demonstrates the feasibility of using the red algae P. cruentum for the preparation of large quantities
Acknowledgements
This research was supported by the Plan Propio de Investigación from University of Almerı́a and Plan Andaluz de Investigación II, Junta de Andalucı́a.
References (54)
- et al.
Natural pigments from red microalgae for use in foods and cosmetics
Trends Food Sci. Technol.
(1992) - et al.
Chromatographic purification of phycobiliproteins from Spirulina platensis. High-performance liquid chromatographic separation of their alfa and beta subunits
J. Chromatogr. A
(1997) - et al.
C-phycocyanin incorporated into reverse micelles: a fluorescence study
Coll. Surf. B Biointerf.
(2000) - et al.
Chromatographic purification and characterization of B-phycoerythrin from Porphyridium cruentum. Semipreparative HPLC separation and characterization of its subunits
J. Chromatogr. A
(2001) - et al.
Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. liui Zhang et Xia
J. Photochem. Photobiol. B Biol.
(1999) - et al.
R-phycoerythrin from the red alga Gracilaria longa
Phytochemistry
(1994) - et al.
Isolation, crystallization, crystal structure analysis and refinement of constitutive C-phycocyanin from the chromatically adapting cyanobacterium Fremyella diplosiphon at 1'66 D resolution
J. Mol. Biol.
(1991) - et al.
Renewal rate of semicontinuous cultures of the microalga Porphyridium cruentum modifies phycoerythrin, exopolysaccharides and fatty acid productivity
J. Ferment. Bioeng.
(1998) - et al.
Isolation, crystallization, crystal structure analysis and refinement of B-phycoerythrin from the red alga Porphyridium sordidum at 2.2 A resolution
Mol. Biol.
(1992) - et al.
One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis
J. Chromatogr. B
(2000)
Structure and function of phycobilisomes: light harvesting pigment complexes in red and blue-green algae
Int. Rev. Cytol.
Phycobilisome. A macromolecular complex optimized for energy tranfer
Biochim. Biophys. Acta
Phycofluor probes
Trends Biochem. Sci.
Phycoerythrin 566. A fluorescence study
Biochim. Biophys. Acta
A nobel and inexpensive source of allophycocyanin for multicolor flow cytometry
J. Immunol. Methods
Growth evaluation and bioproducts characterization of Calothrix spp
Bioresource Technol.
Phycocyanin from Spirulina spp: influence of processing of biomass on phycocyanin yield, analysis of efficacy of extraction methods and stability studies on phycocyanin
Process Biochem.
Purification of phycobiliproteins from Nostoc spp. by aminohexyl-Sepharose chromatography
J. Biotechnol.
Molecular assembly of proteins and conjugated polymers: toward development of biosensors
Biotechnol. Bioeng.
Elution chromatography
Complementary chromatic adaptation in a filamentous blue-green algae
J. Cell. Biol.
The structure of cyanobacterial phycobilisomes: a model
Arch. Microbiol.
Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture
Biotechnol. Bioeng.
Simple and rapid procedure for analyzing two phycocyanins (C-PC and APC) from Spirulina platensis algae using LPLC and HPLC methods
Annali di Chimica
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