Efficient methods for fast, producible, C-Phycocyanin from Thermosynechococcus elongatus
Keywords:
A620/A280 value, C-PC purification, C-Phycocyanin, Cyanobacteria, Fluorescence Spectra, IEC, Phycobilines, Sucrose Gradient, Thermosynechococcus elongatus
Abstract
This article describes different protocols that enhance the extraction, isolation and purification of phycocyanin from the cyanobacterium, Thermosynechococcus elongatus as well as absorbance and fluorescence spectral characterization. A combination of enzymatic degradation by Lysozyme followed by high pressure showed a mild cell wall destruction except for the composition of thylakoid membrane compared with glass beads. The use of ammonium sulfate precipitation as the first purification step exhibited high efficiency in removing most of the protein contamination. The best purified phycocyanin was obtained after using the second purification step that could be ion exchange chromatography or sucrose gradient. Unexpected results that were not used earlier were obtained by sucrose gradient, where a large amount of highly pure phycocyanin was assembled compared with published methods. An evaluation of C-phycocyanin throughout the series steps of isolation and purification was achieved by using absorbance and 77K fluorescence spectral analysis. Besides a spectroscopical evaluation, SDS-PAGE, productivity, and A620/A280 values pointed to the purity and structural preservation of a purified complex. Compared with published methods, the existing method not only reduces purification time but also enhances the productivity of phycocyanin in its native structure.
The optimization of each purification step presented different purified phycocyanin levels; hence, it could be used not only by microbiologists but also by other researchers such as physicians and industrial applicants. In addition, this method could be used as a model for all cyanobacterial species and could be also used for Rhodophytes with some modifications.
References
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Bennett A and Bogorad L. 1973. Complementary chromatic adaptation in a filamentous blue-green alga.
J Cell Biol., 58(2):419-35
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Boussiba S and Richmond AE. 1979. Isolation and characterization of phycocyanins from the blue-green alga Spirulina platensis. Arch Microbiol., 120(2):155– 159.
Bryant DA, Guglielmi G, Tandeau de marsac N, Castets AM and Cohen-Bazire G. 1979. The structure of cyanobacterial phycobilisomes: a model. Arch Microbiol., 123(2):113–127.
Contreras-Martel C, Matamala A, Bruna C, Poo- Caamaño G, Almonacid D, Figueroa M, Martínez-
Oyanedel J and Bunster M. 2007. The structure at 2 Å resolution of phycocyanin from Gracilaria chilensis and the energy transfer network in a PC–PC complex. Biophys Chem., 125(2-3):388–396.
El-Mohsnawy E, Kopczak MJ, Schlodder E, Nowaczyk M, Meyer HE, Warscheid B, Karapetyan NV and Rögner M. 2010. Structure and function of intact photosystem 1 monomers from the cyanobacterium Thermosynechococcus elongatus. Biochemistry. 15; 49(23):4740-4751.
El-Mohsnawy E. 2013. Purification, Characterization, and Activity Evaluation of Allophycocyanin from
Thermosynechococcus elongatus. Life Science Journal. 10(4): 3754-3761. 503.
Ferreira, KN, Iverson, TM, Maghlaoui, K, Barber, J and Iwata S. 2004. Architecture of the photosynthetic oxygen-evolving center. Science. 303 (5665):1831-1838.
Frankenberg N, Moser J and Jahn D. 2003. Bacterial heme biosynthesis and its biotechnological application. Appl Microbiol Biotechnol., 63(2):115-127.
Frankenberg N and Lagarias JC. 2003. Phycocyanobilin: Ferredoxin Oxidoreductase of
Anabaena sp. PCC 7120: Biochemical and spectroscopic characterization. The Journal of Biological Chemistry. 278(11): 9219-9226. DOI 10.1074/jbc.M211643200.
Gan X, Tang X, Shi C, Wang B, Cao Y and Zhao L. 2004. Preparation and regeneration of spheroplasts from Arthrospira platensis (Spirulina). J Appl Phycol., 16 (6):513–517.
Gupta A and Sainis JK. 2010. Isolation of C-phycocyanin from Synechococcus sp., (Anacystis
nidulans BD1). J Appl Phycol., 22(3):231–233.
Herrera A, Boussiba S, Napoleone V and Hohlberg A. 1989. Recovery of C-phycocyanin from the
cyanobacterium Spirulina maxima. J Appl Phycol., 1 (4):325–331.
Ichimura M, Kato S, Tsuneyama K, Matsutake S, Kamogawa M, Hirao E, Miyata A, Mori S,
Yamaguchi N, Suruga K and Omagari K. 2013. Phycocyanin prevents hypertension and low serum
adiponectin level in a rat model of metabolic syndrome. Nutr Res., 33(5): 397-405.
Jordan P, Fromme P, Witt HT, Klukas O, Saenger W and Krauss N. 2001. Threedimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature. 21 411(6840):909-917.
Katoh H, Hagino N, Grossmann AR and Ogawa T. 2001. Genes essential to iron transport in the
cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol., 183(9): 2779-2784.
Kubota H, Sakurai I, Katayama K, Mizusawa N, Ohashi S, Kobayashi M, Zhang P, Aro EM and
Wada H. 2010. Purification and characterization of photosystem I complex from Synechocystis sp. PCC
6803 by expressing histidine-tagged subunits. Biochim Biophys Acta. 1797(1):98-105.
Kurisu G, Zhang H, Smith JL and Cramer WA. 2003. Structure of the cytochrome b6f complex of
oxygenic photosynthesis: Tuning the cavity. Science. 302(5647):1009-1014.
Lawrenz E, Fedewa EJ and Richardson TL. 2011. Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts. J Appl Phycol., 23(5):865–871.
Li B, Chu X, Gao M and Li W. 2010. Apoptotic mechanism of MCF-7 breast cells in vivo and in vitro
induced by photodynamic therapy with C-phycocyanin. Acta Biochimica et Biophysica Sinica. Sin (Shanghai). 42 (1):80-89.
Loll B, Kern J, Saenger W, Zouni A and Biesiadka J. 2005. Towards complete cofactor arrangement in the 3.0A resolution structure of photosystem II. Nature. 438 (7070):1040-4.
Marín-Prida J, Pavón-Fuentes N, Llópiz-Arzuaga A, Fernández-Massó JR, Delgado-Roche L, Mendoza-Marí Y, Santana SP, Cruz-Ramírez A, Valenzuela- Silva C, Nazábal-Gálvez M, Cintado-Benítez A, Pardo-Andreu GL, Polentarutti N, Riva F, Pentón- Arias E and Pentón-Rol G. 2013. Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats. Toxicol Appl
Pharmacol.,. 1; 272(1):49-60.
Minkova KM, Tchernov AA, Tchorbadjieva MI, Fournadjieva ST, Antova RE and Busheva MCh.
2003. Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J Biotechnol., 102(1):55–59.
Niu JF, Wang GC, Lin XZ and Zhou BC. 2007. Large -scale recovery of C-phycocyanin from Spirulina
platensis using expanded bed adsorption chromatography. J Chromatogr B. 850(1-2): 267–276.
Patil G and Raghavarao KSMS. 2007. Aqueous two phase extraction for purification of C-phycocyanin.
Biochem Eng J., 34(2):156–164.
Pleonsil P and Suwanwong Y. 2013. An in vitro study of c-phycocyanin activity on protection of DNA and human erythrocyte membrane from oxidative damage. Journal of Chemical and Pharmaceutical Research. 5 (5):332-336 .
Rippka R, Deruelles J, Waterbury JB, Herdman M and Stanier RY. 1979. Generic assignments, strain
histories and properties of pure cultures of cyanobacteria. J Gen Microbiology. 111(1):1–61.
Rito-Palomares M, Nuñez L and Amador D. 2001. Practical application of aqueous two phase systems for the development of a prototype process for cphycocyanin recovery from Spirulina maxima. J Chem
Techn Biotechnol., 76(12):1273–1280.
Rögner M, Boekema EJ and Barber J. 1996. How does photosystem 2 split water? The structural basis of efficient energy conversion. Trends Biochem Sci., 21 (2):44-9.
Santiago-Santos MC, Ponce-Noyola T, Olvera- Ramirez R, Ortega-Lopez J and Canizares-
Villanueva RO. 2004. Extraction and purification of phycocyanin from Calothrix sp. Process Biochemistry.
39(12): 2047-2052.
Schägger, H and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analyt Biochem., 166(2): 368-379.
Schlodder E, Shubin VV, El-Mohsnawy E, Rögner M and Karapetyan NV. 2007. Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus. Biochim Biophys Acta. 2007 (6):732-741.
Schopf JW. 2000. The fossil record: tracing the roots of the cyanobacterial lineage. In: Whitton BA, Potts M (eds) The ecology of cyanobacteria: Their Diversity in Time and Space. Kluwer, Dordrecht. p 13–35.
Song W, Zhao C and Wang S. 2013. A Large-Scale Preparation Method of High Purity CPhycocyanin.
International Journal of Bioscience, Biochemistry and Bioinformatics. 3(4):293-297.
Soni B, Trivedi U and Madamwar D. 2008. A novel method of single step hydrophobic interaction
chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for
antioxidant property. Bioresour Technol., 99(1):188– 194.
Sonoike K and Katoh S. 1989. Simple estimation of the differential absorption coefficient of P-700 in detergenttreated preparations. Biochim Biophys Acta. 976(2- 3):210–213.
Stec B, Troxler RF and Teeter MM. 1999. Crystal structure of C-phycocyanin from Cyanidium caldarium
provides a new perspective in on phycobilisome assembly. Biophys J., 76(6): 2912–2921.
Thangam R, Suresh V, Asenath Princy W, Rajkumar M, Senthilkumar N, Gunasekaran P, Rengasamy R, Anbazhagan C, Kaveri K and Kannan S. 2013. C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest. Food Chem., 140(1-2):262-72.
Troxler RF, Ehrhardt MM, Brown-Mason AS and Offner GD. 1981. Primary structure of phycocyanin
from the unicellular rhodophyte Cyanidium caldarium. II. Complete amino acid sequence of the beta subunit. J Biol Chem., 256(23):12176–12184.
Zouni A, Witt HT, Kern J, Fromme P, Krauss N, Saenger W and Orth P. 2001. Crystal structure of
photosystem II from Synechococcus elongatus at 3.8 A resolution. Nature. 409(6821):739-43.
Adir N. 2005. Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant. Photosynth. Res., 85(1): 15–32.
Bennett A and Bogorad L. 1973. Complementary chromatic adaptation in a filamentous blue-green alga.
J Cell Biol., 58(2):419-35
Bhaskar SU, Gopalaswamy G and Raghu R. 2005. A simple method for efficient extraction and purification of C-phycocyanin from Spirulina platensis Geitler. Indian J Exp Biol., 43(3):277-279.
Boussiba S and Richmond AE. 1979. Isolation and characterization of phycocyanins from the blue-green alga Spirulina platensis. Arch Microbiol., 120(2):155– 159.
Bryant DA, Guglielmi G, Tandeau de marsac N, Castets AM and Cohen-Bazire G. 1979. The structure of cyanobacterial phycobilisomes: a model. Arch Microbiol., 123(2):113–127.
Contreras-Martel C, Matamala A, Bruna C, Poo- Caamaño G, Almonacid D, Figueroa M, Martínez-
Oyanedel J and Bunster M. 2007. The structure at 2 Å resolution of phycocyanin from Gracilaria chilensis and the energy transfer network in a PC–PC complex. Biophys Chem., 125(2-3):388–396.
El-Mohsnawy E, Kopczak MJ, Schlodder E, Nowaczyk M, Meyer HE, Warscheid B, Karapetyan NV and Rögner M. 2010. Structure and function of intact photosystem 1 monomers from the cyanobacterium Thermosynechococcus elongatus. Biochemistry. 15; 49(23):4740-4751.
El-Mohsnawy E. 2013. Purification, Characterization, and Activity Evaluation of Allophycocyanin from
Thermosynechococcus elongatus. Life Science Journal. 10(4): 3754-3761. 503.
Ferreira, KN, Iverson, TM, Maghlaoui, K, Barber, J and Iwata S. 2004. Architecture of the photosynthetic oxygen-evolving center. Science. 303 (5665):1831-1838.
Frankenberg N, Moser J and Jahn D. 2003. Bacterial heme biosynthesis and its biotechnological application. Appl Microbiol Biotechnol., 63(2):115-127.
Frankenberg N and Lagarias JC. 2003. Phycocyanobilin: Ferredoxin Oxidoreductase of
Anabaena sp. PCC 7120: Biochemical and spectroscopic characterization. The Journal of Biological Chemistry. 278(11): 9219-9226. DOI 10.1074/jbc.M211643200.
Gan X, Tang X, Shi C, Wang B, Cao Y and Zhao L. 2004. Preparation and regeneration of spheroplasts from Arthrospira platensis (Spirulina). J Appl Phycol., 16 (6):513–517.
Gupta A and Sainis JK. 2010. Isolation of C-phycocyanin from Synechococcus sp., (Anacystis
nidulans BD1). J Appl Phycol., 22(3):231–233.
Herrera A, Boussiba S, Napoleone V and Hohlberg A. 1989. Recovery of C-phycocyanin from the
cyanobacterium Spirulina maxima. J Appl Phycol., 1 (4):325–331.
Ichimura M, Kato S, Tsuneyama K, Matsutake S, Kamogawa M, Hirao E, Miyata A, Mori S,
Yamaguchi N, Suruga K and Omagari K. 2013. Phycocyanin prevents hypertension and low serum
adiponectin level in a rat model of metabolic syndrome. Nutr Res., 33(5): 397-405.
Jordan P, Fromme P, Witt HT, Klukas O, Saenger W and Krauss N. 2001. Threedimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature. 21 411(6840):909-917.
Katoh H, Hagino N, Grossmann AR and Ogawa T. 2001. Genes essential to iron transport in the
cyanobacterium Synechocystis sp. strain PCC 6803. J Bacteriol., 183(9): 2779-2784.
Kubota H, Sakurai I, Katayama K, Mizusawa N, Ohashi S, Kobayashi M, Zhang P, Aro EM and
Wada H. 2010. Purification and characterization of photosystem I complex from Synechocystis sp. PCC
6803 by expressing histidine-tagged subunits. Biochim Biophys Acta. 1797(1):98-105.
Kurisu G, Zhang H, Smith JL and Cramer WA. 2003. Structure of the cytochrome b6f complex of
oxygenic photosynthesis: Tuning the cavity. Science. 302(5647):1009-1014.
Lawrenz E, Fedewa EJ and Richardson TL. 2011. Extraction protocols for the quantification of phycobilins in aqueous phytoplankton extracts. J Appl Phycol., 23(5):865–871.
Li B, Chu X, Gao M and Li W. 2010. Apoptotic mechanism of MCF-7 breast cells in vivo and in vitro
induced by photodynamic therapy with C-phycocyanin. Acta Biochimica et Biophysica Sinica. Sin (Shanghai). 42 (1):80-89.
Loll B, Kern J, Saenger W, Zouni A and Biesiadka J. 2005. Towards complete cofactor arrangement in the 3.0A resolution structure of photosystem II. Nature. 438 (7070):1040-4.
Marín-Prida J, Pavón-Fuentes N, Llópiz-Arzuaga A, Fernández-Massó JR, Delgado-Roche L, Mendoza-Marí Y, Santana SP, Cruz-Ramírez A, Valenzuela- Silva C, Nazábal-Gálvez M, Cintado-Benítez A, Pardo-Andreu GL, Polentarutti N, Riva F, Pentón- Arias E and Pentón-Rol G. 2013. Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats. Toxicol Appl
Pharmacol.,. 1; 272(1):49-60.
Minkova KM, Tchernov AA, Tchorbadjieva MI, Fournadjieva ST, Antova RE and Busheva MCh.
2003. Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. J Biotechnol., 102(1):55–59.
Niu JF, Wang GC, Lin XZ and Zhou BC. 2007. Large -scale recovery of C-phycocyanin from Spirulina
platensis using expanded bed adsorption chromatography. J Chromatogr B. 850(1-2): 267–276.
Patil G and Raghavarao KSMS. 2007. Aqueous two phase extraction for purification of C-phycocyanin.
Biochem Eng J., 34(2):156–164.
Pleonsil P and Suwanwong Y. 2013. An in vitro study of c-phycocyanin activity on protection of DNA and human erythrocyte membrane from oxidative damage. Journal of Chemical and Pharmaceutical Research. 5 (5):332-336 .
Rippka R, Deruelles J, Waterbury JB, Herdman M and Stanier RY. 1979. Generic assignments, strain
histories and properties of pure cultures of cyanobacteria. J Gen Microbiology. 111(1):1–61.
Rito-Palomares M, Nuñez L and Amador D. 2001. Practical application of aqueous two phase systems for the development of a prototype process for cphycocyanin recovery from Spirulina maxima. J Chem
Techn Biotechnol., 76(12):1273–1280.
Rögner M, Boekema EJ and Barber J. 1996. How does photosystem 2 split water? The structural basis of efficient energy conversion. Trends Biochem Sci., 21 (2):44-9.
Santiago-Santos MC, Ponce-Noyola T, Olvera- Ramirez R, Ortega-Lopez J and Canizares-
Villanueva RO. 2004. Extraction and purification of phycocyanin from Calothrix sp. Process Biochemistry.
39(12): 2047-2052.
Schägger, H and von Jagow, G. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analyt Biochem., 166(2): 368-379.
Schlodder E, Shubin VV, El-Mohsnawy E, Rögner M and Karapetyan NV. 2007. Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus. Biochim Biophys Acta. 2007 (6):732-741.
Schopf JW. 2000. The fossil record: tracing the roots of the cyanobacterial lineage. In: Whitton BA, Potts M (eds) The ecology of cyanobacteria: Their Diversity in Time and Space. Kluwer, Dordrecht. p 13–35.
Song W, Zhao C and Wang S. 2013. A Large-Scale Preparation Method of High Purity CPhycocyanin.
International Journal of Bioscience, Biochemistry and Bioinformatics. 3(4):293-297.
Soni B, Trivedi U and Madamwar D. 2008. A novel method of single step hydrophobic interaction
chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for
antioxidant property. Bioresour Technol., 99(1):188– 194.
Sonoike K and Katoh S. 1989. Simple estimation of the differential absorption coefficient of P-700 in detergenttreated preparations. Biochim Biophys Acta. 976(2- 3):210–213.
Stec B, Troxler RF and Teeter MM. 1999. Crystal structure of C-phycocyanin from Cyanidium caldarium
provides a new perspective in on phycobilisome assembly. Biophys J., 76(6): 2912–2921.
Thangam R, Suresh V, Asenath Princy W, Rajkumar M, Senthilkumar N, Gunasekaran P, Rengasamy R, Anbazhagan C, Kaveri K and Kannan S. 2013. C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest. Food Chem., 140(1-2):262-72.
Troxler RF, Ehrhardt MM, Brown-Mason AS and Offner GD. 1981. Primary structure of phycocyanin
from the unicellular rhodophyte Cyanidium caldarium. II. Complete amino acid sequence of the beta subunit. J Biol Chem., 256(23):12176–12184.
Zouni A, Witt HT, Kern J, Fromme P, Krauss N, Saenger W and Orth P. 2001. Crystal structure of
photosystem II from Synechococcus elongatus at 3.8 A resolution. Nature. 409(6821):739-43.
Published
2014-02-10
How to Cite
Eithar, E.-M. (2014). Efficient methods for fast, producible, C-Phycocyanin from Thermosynechococcus elongatus. Journal of Research in Biology, 3(8), 1132-1146. Retrieved from https://ojs.jresearchbiology.com/ojs1/index.php/jrb/article/view/326
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