Large scale preparation of phycobiliproteins from Porphyra yezoensis using co-precipitation with ammonium sulfate
ABSTRACT
The purpose of this study was to extract phyco-biliproteins (PBP) from Porphyra yezoensis using an optimized procedure and further establish a large scale process for protein production. According to our previous experiences on the extraction of PBP, salting out methods, e.g. ammonium sulfate precipitation, worked more efficiently than isoelectric precipitation, differential centrifugation or ultrafiltration. Thus, we chose ammonium sulfate to coprecipitate PBP in crude solution. After four times of precipitation followed by one time of high speed centrifugation, the maximum purity of crude phycoerythrin and phycocyanin reached 1.94 (A565/A280) and 0.85 (A615/A280), with a yield of 0.50% and 0.37%, respectively. A total of 0.94 mg phycoerythrin and 0.54 mg phycocyanin with purity of more than 3.2 were obtained from 1 g dried P. yezoensis after additional chromatography. We further scaled up the frozen dried P. yezoensis from 20 g to 400 g, with 1295 mg phycoerythrin and 593 mg phycocyanin obtained.
The purpose of this study was to extract phyco-biliproteins (PBP) from Porphyra yezoensis using an optimized procedure and further establish a large scale process for protein production. According to our previous experiences on the extraction of PBP, salting out methods, e.g. ammonium sulfate precipitation, worked more efficiently than isoelectric precipitation, differential centrifugation or ultrafiltration. Thus, we chose ammonium sulfate to coprecipitate PBP in crude solution. After four times of precipitation followed by one time of high speed centrifugation, the maximum purity of crude phycoerythrin and phycocyanin reached 1.94 (A565/A280) and 0.85 (A615/A280), with a yield of 0.50% and 0.37%, respectively. A total of 0.94 mg phycoerythrin and 0.54 mg phycocyanin with purity of more than 3.2 were obtained from 1 g dried P. yezoensis after additional chromatography. We further scaled up the frozen dried P. yezoensis from 20 g to 400 g, with 1295 mg phycoerythrin and 593 mg phycocyanin obtained.
KEYWORDS
Porphyra yezoensis; Phycoerythrin; Phycocyanin; Ammonium Sulfate Precipitation; Hydroxylapatite
Porphyra yezoensis; Phycoerythrin; Phycocyanin; Ammonium Sulfate Precipitation; Hydroxylapatite
Cite this paper
Cai, C. , Li, C. , Wu, S. , Wang, Q. , Guo, Z. and He, P. (2012) Large scale preparation of phycobiliproteins from Porphyra yezoensis using co-precipitation with ammonium sulfate. Natural Science, 4, 536-543. doi: 10.4236/ns.2012.48071.
Cai, C. , Li, C. , Wu, S. , Wang, Q. , Guo, Z. and He, P. (2012) Large scale preparation of phycobiliproteins from Porphyra yezoensis using co-precipitation with ammonium sulfate. Natural Science, 4, 536-543. doi: 10.4236/ns.2012.48071.
References
[1] Liu, L.N., Chen, X.L., Zhang, X.Y., Zhang, Y.Z. and Zhou, B.C. (2005) One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata. Journal of Biotechnology, 116, 91-100. doi:10.1016/j.jbiotec.2004.09.017 [2] Dufosséa, L., Galaupa, P., Yaronb, A., Aradb, S.M., Blancc, P., Murthyd, K.N.C. and Ravishankard, G.A. (2005) Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends in Food Science & Technology, 16, 389-406. doi:10.1016/j.tifs.2005.02.006 [3] Santiago-Santos, M.C., Ponce-Noyola, T., Olvera-RamIrez, R., Ortega-López, J. and Canizares-Villanueva, R.O. (2004) Extraction and purification of phycocyanin from Calothrix sp. Process Biochemistry, 39, 2047-2052. doi:10.1016/j.procbio.2003.10.007 [4] Kronick, M.N. and Grossman, P.D. (1983) Immunoassay techniques with fluores-cent phycobiliprotein conjugates. Clinical Chemistry, 29, 1582-1586. [5] Sekar, S. and Chandramohan, M. (2008) Phycobiliproteins as a commodity: Trends in applied research, patents and commercialization. Journal of Applied Phycology, 20, 113-136. doi:10.1007/s10811-007-9188-1 [6] Benedetti, S., Benvenuti, F., Pagliarani, S., Francogli, S., Scoglio, S. and Canestrari, F. (2004) Antioxidant properties of a novel phyco-cyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sciences, 75, 2353- 2362. doi:10.1016/j.lfs.2004.06.004 [7] Cai, X.H., Zheng, S., He, L.M. and Jiang, J.L. (1995) Study on promotion of laser therapy for carcinoma using phycocyanin from Spirulina. Chinese Journal of Marine Drugs, 12, 290-291. [8] Nagaoka, S., Shimizu, K., Kaneko, H., Shibayama, F., Morikawa, K., Kanamaru, Y., Otsuka, A., Hirahashi, T. and Kato, T. (2005) A novel protein C-phycocyanin plays a crucial role in the hypocholes-terolemic action of Spirulina platensis concentrate in rats. The Journal of Nutrition, 135, 2425-2430. [9] Shih, C.M., Cheng, S.N., Chih-Shung Wong Kuo, Y.L. and Chou, T.C. (2009) An-tiinflammatory and antihyperalgesic activity of C-phycocyanin. Anesthesia & Analgesia, 108, 1303-1310. doi:10.1213/ane.0b013e318193e919 [10] Burrows, S.M. Patel, P. and Pappas, D. (2009) Light tolerance of R-phycoerythrin and a tandem conjugate observed by single molecule recrossing events. Applied Spectroscopy, 63, 709-715. doi:10.1366/000370209788559737 [11] Womick, J.M. and Moran, A.M. (2009) Nature of excited states and relaxation mechanisms in C-phycocyanin. The Journal of Physical Chemistry B, 113, 15771-15782. doi:10.1021/jp908093x [12] Sun, L., Wang, S., Gong, X., Zhao, M., Fu, X. and Wang, L. (2009) Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica. Protein Expression and Purification, 64, 146-154. doi:10.1016/j.pep.2008.09.013 [13] Padgett, M.P. and Krogmann, D.W. (1987) Large scale preparation of pure phycobiliproteins. Photosynthesis Research, 11, 225-235. doi:10.1007/BF00055062 [14] Wang, G.C. (2002) Isolation and purification of phycoerythrin from red alga Gracilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatography. Chromatographia, 56, 509-513. [15] Wang, G.C., Sun, H.B., Fan, X. and Tseng, C.K. (2002) Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method. Journal of Integrat Plant Biology, 44, 541-546. [16] Niu, J.F., Wang, G.C., Zhou, B.C., Lin, X.Z. and Chen, C.S. (2007) Purification of R-phycoerythrin from Porphyra haitanensis (Bangiales, Rhodophyta) using expanded-bed absorption. Journal of Phycology, 43, 1339- 1347. [17] Keiller, D.R., Whitelam, G.C. and Smith, H. (1988) Polyclonal antibodies raised to phyco-cyanins contain components specific for the red-absorbing form of phytochrome. Planta, 176, 391-398. doi:10.1007/BF00395420 [18] Cai, Y.A., Murphy, J.T., Wedemayer, G.J. and Glazer, A.N. (2001) Recombinant phycobili-proteins: Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains. Analytical Biochemistry, 290, 186-204. [19] St'astná, M., Radko, S. and Chrambach, A. (2000) Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters. Electrophoresis, 21, 985-992. [20] Viskari, P.J. and Colyer, C.L. (2002) Separation and quantitation of phyco-biliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection. Journal of Chromato-graphy A, 972, 269-276. doi:10.1016/S0021-9673(02)01085-3 [21] Viskaria, P.J. and Colyer, C.L. (2003) Rapid extraction of phycobiliproteins from cultured cyanobacteria samples. Analytical Biochemistry, 319, 263-271. doi:10.1016/S0003-2697(03)00294-X [22] Ranjitha, K. and Kaushik, B.D. (2005) Purification of phycobiliproteins from Nostoc muscorum. Journal of Scientific and Industrial Research, 64, 372-375. [23] Niu, J.F., Chen, Z.F., Wang, G.C. and Zhou, B.C. (2009) Purification of phycoerythrin from Porphyra yezoensis Ueda (Bangiales, Rhodophyta) using expanded bed absorption. Journal of Applied Phycology, 22, 25-31. [24] Siegelman, H. and Kycia, J. (1978) Algal bili-proteins: Handbook of phycological method. Cambridge University Press, Cambridge, 71-79. [25] Zheng, J., Gao, Y.H., Wang, W.X. and Huang, S.Y. (2003) Studies on crude extraction methods of phycoerythrin from Bangia fusco-purpurea and denaturalization mechanism of phycobiliprotein in different irradiation conditions. Journal of Xiamen University (Natural Science), 42, 117-122. [26] Peng, W., Shang, S., Liu, G., Fu, Y. and Wu, Q. (1999) Extraction of phycobiliprotein from Spirulina platensis (Sp,-D). Food Science, 20, 48-49. [27] Hu, Y., Hu, H., Li, Y. and Geng, Y. (2002) Study on mass production of phycobilins from PC rich strain of Spirulina platensis. Journal of Wuhan Botanical Research, 20, 299-302. [28] Cohen, Z., Reungjitchachawali, M., Siangdung, W. and Tanticharoen, M. (1993) Production and partial purification of γ-linolenic acid and some pigments from Spirulina platensis. Journal of Applied Phycology, 5, 109-115. doi:10.1007/BF02182428 [29] Minkova, K., Tchorbadjieva, M., Tchernov, A., Stojanova, M., Gigova, L. and Busheva, M. (2007) Improved procedure for separation and purification of Arthronema africanum phycobiliproteins. Biotechnology Letters, 29, 647- 651. doi:10.1007/s10529-006-9274-5 [30] Tcheruov, A.A., Minkova, K.M., Georgiev, D.I. and Houbavenska, N.B. (1993) Method for B-phycoerythrin purification from Porphyridium cruentum. Biotechnology Techniques, 7, 853-858. doi:10.1007/BF00156361 [31] Tchernova, A.A., Minkovab, K.M., Houbavenskab, N.B. and Kovachevac, N.G. (1999) Puri-fication of phycobiliproteins from Nostoc sp. by aminohex-yl-Sepharose chromatography. Journal of Biotechnology, 69, 69-73. doi:10.1016/S0168-1656(98)00206-5 [32] Minkova, K.M., Tchernov, A.A., Tchorbadjieva, M.I., Fournadjieva, S.T., Antova, R.E. and Busheva, M.C. (2003) Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. Journal of Biotechnology, 102, 55-59. doi:10.1016/S0168-1656(03)00004-X [33] Reisa, A., Mendesb, A., Lobo-Fernandesa, H., Empisb, J.A. and Novaisb, J.M. (1998) Production, extraction and purification of phycobili-proteins from Nostoc sp. Bioresource Technology, 66, 181-187. doi:10.1016/S0960-8524(98)00064-9 [34] Sudarsanan, K. and Young, R.A. (1969) Significant precision in crystal structural details. Holly Springs hydroxyapatite. Acta Crystallographica Section B Structural Crystallography and Crystal Chemistry, 25, 1534-1543. doi:10.1107/S0567740869004298 [35] Pu, Y., Jiang, L. and Zeng, F. (1986) Preparation of allo-phycocyanin and studies on the characteristics of allo- phycocyanin from Porphyra haitanesis. Science Bulletin, 4, 272-274. [36] Grabowski, J. and Gantt, E. (1978) Photophysical properties of phycobiliproteins from phycobilisomes: Fluorescence lifetimes, quantum yields, and polarization spectra. Photochemstry and Photobiology, 28, 39-45. doi:10.1111/j.1751-1097.1978.tb06927.x [37] He, P.M., Xu, S., Zhang, H., Wen, S., Dai, Y., Lin, S. and Yarish, C. (2008) Bio-remediation efficiency in the removal of dissolved inorganic nutrients by the red seaweed, Porphyra yezoensis, cultivated in the open sea. Water Research, 42, 1281-1289. doi:10.1016/j.watres.2007.09.023
[1] Liu, L.N., Chen, X.L., Zhang, X.Y., Zhang, Y.Z. and Zhou, B.C. (2005) One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata. Journal of Biotechnology, 116, 91-100. doi:10.1016/j.jbiotec.2004.09.017 [2] Dufosséa, L., Galaupa, P., Yaronb, A., Aradb, S.M., Blancc, P., Murthyd, K.N.C. and Ravishankard, G.A. (2005) Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends in Food Science & Technology, 16, 389-406. doi:10.1016/j.tifs.2005.02.006 [3] Santiago-Santos, M.C., Ponce-Noyola, T., Olvera-RamIrez, R., Ortega-López, J. and Canizares-Villanueva, R.O. (2004) Extraction and purification of phycocyanin from Calothrix sp. Process Biochemistry, 39, 2047-2052. doi:10.1016/j.procbio.2003.10.007 [4] Kronick, M.N. and Grossman, P.D. (1983) Immunoassay techniques with fluores-cent phycobiliprotein conjugates. Clinical Chemistry, 29, 1582-1586. [5] Sekar, S. and Chandramohan, M. (2008) Phycobiliproteins as a commodity: Trends in applied research, patents and commercialization. Journal of Applied Phycology, 20, 113-136. doi:10.1007/s10811-007-9188-1 [6] Benedetti, S., Benvenuti, F., Pagliarani, S., Francogli, S., Scoglio, S. and Canestrari, F. (2004) Antioxidant properties of a novel phyco-cyanin extract from the blue-green alga Aphanizomenon flos-aquae. Life Sciences, 75, 2353- 2362. doi:10.1016/j.lfs.2004.06.004 [7] Cai, X.H., Zheng, S., He, L.M. and Jiang, J.L. (1995) Study on promotion of laser therapy for carcinoma using phycocyanin from Spirulina. Chinese Journal of Marine Drugs, 12, 290-291. [8] Nagaoka, S., Shimizu, K., Kaneko, H., Shibayama, F., Morikawa, K., Kanamaru, Y., Otsuka, A., Hirahashi, T. and Kato, T. (2005) A novel protein C-phycocyanin plays a crucial role in the hypocholes-terolemic action of Spirulina platensis concentrate in rats. The Journal of Nutrition, 135, 2425-2430. [9] Shih, C.M., Cheng, S.N., Chih-Shung Wong Kuo, Y.L. and Chou, T.C. (2009) An-tiinflammatory and antihyperalgesic activity of C-phycocyanin. Anesthesia & Analgesia, 108, 1303-1310. doi:10.1213/ane.0b013e318193e919 [10] Burrows, S.M. Patel, P. and Pappas, D. (2009) Light tolerance of R-phycoerythrin and a tandem conjugate observed by single molecule recrossing events. Applied Spectroscopy, 63, 709-715. doi:10.1366/000370209788559737 [11] Womick, J.M. and Moran, A.M. (2009) Nature of excited states and relaxation mechanisms in C-phycocyanin. The Journal of Physical Chemistry B, 113, 15771-15782. doi:10.1021/jp908093x [12] Sun, L., Wang, S., Gong, X., Zhao, M., Fu, X. and Wang, L. (2009) Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica. Protein Expression and Purification, 64, 146-154. doi:10.1016/j.pep.2008.09.013 [13] Padgett, M.P. and Krogmann, D.W. (1987) Large scale preparation of pure phycobiliproteins. Photosynthesis Research, 11, 225-235. doi:10.1007/BF00055062 [14] Wang, G.C. (2002) Isolation and purification of phycoerythrin from red alga Gracilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatography. Chromatographia, 56, 509-513. [15] Wang, G.C., Sun, H.B., Fan, X. and Tseng, C.K. (2002) Large-scale isolation and purification of R-phycoerythrin from red alga Palmaria palmata using the expanded bed adsorption method. Journal of Integrat Plant Biology, 44, 541-546. [16] Niu, J.F., Wang, G.C., Zhou, B.C., Lin, X.Z. and Chen, C.S. (2007) Purification of R-phycoerythrin from Porphyra haitanensis (Bangiales, Rhodophyta) using expanded-bed absorption. Journal of Phycology, 43, 1339- 1347. [17] Keiller, D.R., Whitelam, G.C. and Smith, H. (1988) Polyclonal antibodies raised to phyco-cyanins contain components specific for the red-absorbing form of phytochrome. Planta, 176, 391-398. doi:10.1007/BF00395420 [18] Cai, Y.A., Murphy, J.T., Wedemayer, G.J. and Glazer, A.N. (2001) Recombinant phycobili-proteins: Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains. Analytical Biochemistry, 290, 186-204. [19] St'astná, M., Radko, S. and Chrambach, A. (2000) Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters. Electrophoresis, 21, 985-992. [20] Viskari, P.J. and Colyer, C.L. (2002) Separation and quantitation of phyco-biliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection. Journal of Chromato-graphy A, 972, 269-276. doi:10.1016/S0021-9673(02)01085-3 [21] Viskaria, P.J. and Colyer, C.L. (2003) Rapid extraction of phycobiliproteins from cultured cyanobacteria samples. Analytical Biochemistry, 319, 263-271. doi:10.1016/S0003-2697(03)00294-X [22] Ranjitha, K. and Kaushik, B.D. (2005) Purification of phycobiliproteins from Nostoc muscorum. Journal of Scientific and Industrial Research, 64, 372-375. [23] Niu, J.F., Chen, Z.F., Wang, G.C. and Zhou, B.C. (2009) Purification of phycoerythrin from Porphyra yezoensis Ueda (Bangiales, Rhodophyta) using expanded bed absorption. Journal of Applied Phycology, 22, 25-31. [24] Siegelman, H. and Kycia, J. (1978) Algal bili-proteins: Handbook of phycological method. Cambridge University Press, Cambridge, 71-79. [25] Zheng, J., Gao, Y.H., Wang, W.X. and Huang, S.Y. (2003) Studies on crude extraction methods of phycoerythrin from Bangia fusco-purpurea and denaturalization mechanism of phycobiliprotein in different irradiation conditions. Journal of Xiamen University (Natural Science), 42, 117-122. [26] Peng, W., Shang, S., Liu, G., Fu, Y. and Wu, Q. (1999) Extraction of phycobiliprotein from Spirulina platensis (Sp,-D). Food Science, 20, 48-49. [27] Hu, Y., Hu, H., Li, Y. and Geng, Y. (2002) Study on mass production of phycobilins from PC rich strain of Spirulina platensis. Journal of Wuhan Botanical Research, 20, 299-302. [28] Cohen, Z., Reungjitchachawali, M., Siangdung, W. and Tanticharoen, M. (1993) Production and partial purification of γ-linolenic acid and some pigments from Spirulina platensis. Journal of Applied Phycology, 5, 109-115. doi:10.1007/BF02182428 [29] Minkova, K., Tchorbadjieva, M., Tchernov, A., Stojanova, M., Gigova, L. and Busheva, M. (2007) Improved procedure for separation and purification of Arthronema africanum phycobiliproteins. Biotechnology Letters, 29, 647- 651. doi:10.1007/s10529-006-9274-5 [30] Tcheruov, A.A., Minkova, K.M., Georgiev, D.I. and Houbavenska, N.B. (1993) Method for B-phycoerythrin purification from Porphyridium cruentum. Biotechnology Techniques, 7, 853-858. doi:10.1007/BF00156361 [31] Tchernova, A.A., Minkovab, K.M., Houbavenskab, N.B. and Kovachevac, N.G. (1999) Puri-fication of phycobiliproteins from Nostoc sp. by aminohex-yl-Sepharose chromatography. Journal of Biotechnology, 69, 69-73. doi:10.1016/S0168-1656(98)00206-5 [32] Minkova, K.M., Tchernov, A.A., Tchorbadjieva, M.I., Fournadjieva, S.T., Antova, R.E. and Busheva, M.C. (2003) Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis. Journal of Biotechnology, 102, 55-59. doi:10.1016/S0168-1656(03)00004-X [33] Reisa, A., Mendesb, A., Lobo-Fernandesa, H., Empisb, J.A. and Novaisb, J.M. (1998) Production, extraction and purification of phycobili-proteins from Nostoc sp. Bioresource Technology, 66, 181-187. doi:10.1016/S0960-8524(98)00064-9 [34] Sudarsanan, K. and Young, R.A. (1969) Significant precision in crystal structural details. Holly Springs hydroxyapatite. Acta Crystallographica Section B Structural Crystallography and Crystal Chemistry, 25, 1534-1543. doi:10.1107/S0567740869004298 [35] Pu, Y., Jiang, L. and Zeng, F. (1986) Preparation of allo-phycocyanin and studies on the characteristics of allo- phycocyanin from Porphyra haitanesis. Science Bulletin, 4, 272-274. [36] Grabowski, J. and Gantt, E. (1978) Photophysical properties of phycobiliproteins from phycobilisomes: Fluorescence lifetimes, quantum yields, and polarization spectra. Photochemstry and Photobiology, 28, 39-45. doi:10.1111/j.1751-1097.1978.tb06927.x [37] He, P.M., Xu, S., Zhang, H., Wen, S., Dai, Y., Lin, S. and Yarish, C. (2008) Bio-remediation efficiency in the removal of dissolved inorganic nutrients by the red seaweed, Porphyra yezoensis, cultivated in the open sea. Water Research, 42, 1281-1289. doi:10.1016/j.watres.2007.09.023