PROCESS TO PURIFY THE COLORED PROTEIN B-PHYCOERYTHRIN PRODUCED BY PORPHYRIDIUM CRUENTUM

  • T. Hernández-Míreles
  • J. Benavides Benavides
  • M. Rito-Palomares
Keywords: aqueous two-phase systems, B-phycoerythrin, Porphyridium cruentum, protein recovery

Abstract

A simplified process for B-phycoerythrin (BPE) purification from Porphyridium cruentum exploiting aqueous twophase systems (ATPS) and isoelectric precipitation was developed. The effect of two different methods of cell disruption upon the release of the intracellular proteins produced by P. cruentum was evaluated. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of BPE released was achieved. The evaluation of partition behaviour of BPE in poly-ethylene-glycol (PEG)-sulphate, PEGdextran and PEG-phosphate ATPS was carried out to determine under which conditions the BPE and contaminants concentrated to opposite phases. ATPS formed with PEG of molecular weight of 1000 g/mol and phosphate proved to be suitable, after isoelectric precipitation at pH 4.0 for the recovery of highly purified BPE (defined as the absorbance ratio of A545nm to A280nm greater than 4.0) with a potential commercial value of $50 USD/mg. An ATPS extraction stage comprising PEG1000 29.5% w/w, phosphate 9.0% w/w, volume ratio (Vr) equal to 1.0, system pH of 7.0, loaded with BPE extract generated by precipitation (equivalent to the 40% w/w of the total extraction system), allowed BPE recovery with a purity of 4.1+0.2 and an overall product yield of 72% (w/w). The purity of BPE from the crude extract increased 5.9 fold after isoelectric precipitation and ATPS. The results reported herein demonstrated the benefits of the practical application of isoelectric precipitation together with ATPS for the
development of a process to purify BPE produced by Porphyridium cruentum.

References

Arad S y Yaron A. (1992). Natural pigments from red microalgae for use in foods and cosmetics. Trends Food Science and Technology 3, 92-97.

Ayyagari M, Pande R, Kamtekar S, Gao H, Marx K, Kumar J, Tripathy S, Akkara J y Kaplan D. (1995). Molecular assembly of proteins and conjugated polymers: Towards development of biosensors. Biotechnology and Bioengineering 45, 116-121.

Benavides J y Rito-Palomares M. (2004). Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum. Journal of Chromatography B 807, 33-38.

Benavides J y Rito-Palomares M. (2005). Potential aqueous two-phase processes for the primary recovery of colored proteins from microbial origin. Engineering in Life Science 5, 259-266.

Bennet A y Bogorad L. (1973). Complementary chromatic adaptation in a filamentous bluegreen algae. Journal of Cell Biology 58, 419-435.

Bermejo R, Alvarez-Pez JM, Acien-Fernandez FG y Molina-Grima E. (2002). Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum. Journal of Biotechnology 93, 73-85.

Bermejo R, Acien FG, Ibáñez MJ, Fernández JM, Molina E y Alvarez-Pez JM. (2003). Preparative purification of b-phycoerythrin from microalga Porphyridium cruentum by expanded-bed adsorption chromatography. Journal of Chromatography B 790,317-325.

Bradford MM. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248-254.

Brass G M F, Simon G W y Lyddiatt A. (2000). Recovery in aqueous two-phase systems of nanoparticulates applied as surrogate mimics for viral gene therapy vectors. Journal of Chromatography B 743, 409-419.

Glatz C E. (1990). Precipitation. In Downstream Processing Biotechnology. Asenjo J. Ed. Marcel Dekker. Nueva York.

Haugland RP. (1996). Handbook of Fluorescent and Research Chemicals. 6th ed. Molecular Probes, Eugene.

He C, Li S, Liu H, Li K y Liu F. (2005). Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt. Journal of Chromatography A, 1082, 143-149.

Koller KP, Wehrmeyer W. y Schneider H. (1977). Isolation and characterization of discshaped phycobilisomes from the red alga Rhodella violacea. Archives of Microbiology 112, 61-67.

Laemmli UK. (1970). Cleavage of strucutral proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Liu LN, Chen XL, Zhang XY y Zhou BC. (2005). One-step chromatography method for efficient separation and purification of Rphycoerythrin from Polysiphonia urceolata. Journal of Biotechnology 116, 91-100.

Market Corporation, 2005, web page: http://www.marketbio.com. (consultada en Octubre 2005).

Qiu J, Madoz-Gurpide J, Misek DE, Kuick R, Brenner DE, Michailids G, Haab BB, Omenn GS, y Hanash S. (2004). Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens. Journal of Proteome Research 3, 261-267.

Ranjitha K y Kaushik BD. (2005). Purification of phycobiliproteins from Nostoc muscorum. Journal of Scientific and Industrial Research 64, 372-375.

Rito-Palomares M, Dale C y Lyddiatt A. (2000a). Generic application of an aqueous two-phase process for protein recovery from animal blood. Process Biochemistry 35 665-673.

Rito-Palomares M, Negrete, Galindo E y Serrano-Carreon L. (2000b). Aroma compounds recovery from micelial cultures in aqueous two-phase processes. Journal of Chromatography, B 743, 403-408.

Rito-Palomares M y Lyddiatt A. (2000) Practical implementation of aqueous two-phase processes for protein recovery from yeast. Journal of Chemical Technology and Biotechnology 75, 632-638.

Rito-Palomares M, Nuñez L y Amador D. (2001). Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima. Journal of Chemical Technology and Biotechnology 76, 1272-1280.

Rito-Palomares M. (2004). Practical application of aqueous two-phase partition to process development for the recovery of biological products. Journal of Chromatography B 807, 3-11.

Ritter, S., Hiller, R. G., Wrench, P. M., Welte, W. y Diederichs, K. (1999). Crystal structure of a phycourobilin-containing phycoerythrin at 1.90-Å resolution. Journal of Structural Biology 126, 86-97.

Swanson RV y Glazer AN. (1990). Separation of phycobiliproteins subunits by reverse-phase high-pressure liquid chromatography. Analytical Biochemistry 188,295-299.

Zaman F, Kusnadi A R y Glatz C E. (1999). Strategies for recombinant protein recovery from canola by precipitation. Biotechnolology Progress 15, 488-492.

Zhu BH, Wang GC y Tseng CK. (2005). Interaction between phycobilisomes from Porphyridium cruentum and thylakoid membranes from Gymmnodinium sp or spinach. Russian Journal of Plant Physiology 52, 288-293.

Zijlstra G M, de Gooijer C D, van der Pol L A y Tramper J. (1996). Design of aqueous twophase systems supporting animal cell growth: a first step towards extractive bioconversion Enzyme Microbial Technology 19, 2-8.
Published
2020-07-15
How to Cite
Hernández-Míreles, T., Benavides, J. B., & Rito-Palomares, M. (2020). PROCESS TO PURIFY THE COLORED PROTEIN B-PHYCOERYTHRIN PRODUCED BY PORPHYRIDIUM CRUENTUM. Revista Mexicana De Ingeniería Química, 5(2), 131-143. Retrieved from http://www.rmiq.org/ojs311/index.php/rmiq/article/view/1903
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