Potential Aqueous Two-Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin By J. Benavides and M. Rito-Palomares* The primary recovery of c-phycocyanin and b-phycoerythrin from Spirulina maxima and Porphyridium cruentum, respec- tively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)-phosphate aqueous two-phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c-phycocyanin and b-phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One-stage processes are proposed for the primary recovery of the colored proteins. PEG1450-phosphate ATPS extraction (volume ratio (V R ) equal to 0.3, tie-line length (TLL) of 34 % w/w and sys- tem pH 7.0) for the recovery of c-phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000-phosphate ATPS extraction (i.e., V R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b-phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the rela- tionship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c-phycocyanin and b-phycoery- thrin from the crude extract increased 3- and 4-fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes. 1 Introduction The urgent need for manufacturers to rapidly and eco- nomically bring new biotechnological products to the mar- ket has oriented research to establish selective and scal- able methods of primary product recovery that integrate effectively with upstream cell cultures. The resulting pro- tocols will have to yield product in a state suitable for vali- dation of polishing, formulation and delivery operations. Currently, biotechnological companies are focusing on the production of high-value products that will have impact in different industrial sectors, such as health, chemistry, food and others. In this context, coloring compounds used in food, cosmetic, detergent and molecular genetics indus- tries are products of great commercial significance [1,2]. The potential production of these substances by microor- ganisms presents a very interesting opportunity for bio- technological processes. Particularly, the production of colored proteins, named phycobiliproteins, from microbial origin represents a very interesting case because both the industrial application and commercial value of these prod- ucts are considerable [1]. The phycobiliproteins are usually found in algae and cyanobacteria as a part of the photo- synthetic system of these microorganisms [3±5]. Two microorganisms that have exhibited great potential for the production of these colorant compounds are Spirulina maxima and Porphyridium cruentum. The commercial value of food grade c-phycocyanin (a blue-colored protein) produced by Spirulina maxima (purity of 0.7, defined as the relationship of 620 nm to 280 nm ab- sorbance) is approximately US$ 0.13 per mg, while that of reactive grade c-phycocyanin (purity of 3.9) varies from US$ 1 to US$ 5 per mg [5]. In contrast, the commercial value of analytical grade c-phycocyanin (purity greater than 4.0) can be as high as US$ 15 per mg [6]. c-Phycocyanin (molecu- lar weight of 44 kDa) is one of the two main biliproteins ob- tained from the photosynthetic systems of Spirulina maxima. It is formed by two sub-units, a and b, with a molecular weigh of 20.5 and 23.5 kDa, respectively, and its isoelectric point has been reported [7] to be around 5.8. In the case of Porphyridium cruentum, b-phycoerythrin (a red-colored protein) is the most valuable of the three main classes of phycoerythrins (B, R and C) on account of its photosynthetic system which offers a wide range of potential commercial applications, such as in natural dyes, food, cosmetics and in the development of biosensors [8, 9]. The commercial value of highly purified b-phycoerythrin (purity greater than 4, de- fined as the relationship of 545 to 280 absorbance) for phar- maceutical or fluorescent uses can be more than US$ 50 per mg [10, 11]. b-Phycoerythrin (molecular weight of 245 kDa) is formed by three subunits, a, b, and c (in a relative molar ratio of 6:6:1) with a molecular weight of 18.0, 18.0, and 29 kDa, respectively [12]. The recovery of these colored proteins from microbial ori- gin has been previously attempted [9, 13, 14]. However, the resulting protocols identified the need to improve the pri- Eng. Life Sci. 2005, 5, No. 3 DOI: 10.1002/elsc.200420073  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 259 ± [*] J. Benavides, M. Rito-Palomares (author to whom correspondence should be addressed: e-mail: mrito@itesm.mx), Centro de Biotecnología, Departamento de Tecnología de Alimentos, Instituto Tecnológico y de Estudios Superiores de Monterrey (ITESM), Ave Eugenio Garza Sada 2501-Sur, Monterrey, NL 64849, Mexico. Eng. Life Sci. Recovery of Colored Proteins Eng. Life Sci.