Purification Of C-phycocyanin Research Articles

Extraction, purification, characterization and bioactivity evaluation of high purity C-phycocyanin from Spirulina sp. NCIM 5143

The study demonstrates an end-to-end downstream process for extraction and purification of C-phycocyanin from Spirulina sp. NCIM 5143. High C-phycocyanin yield of 155.8 mg g−1 DCW, achieved in a single freeze thaw cycle and significantly shorter treatment time of 6.5 h, attributed to the optimized combinatorial process engineering strategy of: screening of suitable biomass condition (wet) and freezing with buffer at an optimized biomass to solvent ratio (1:100). High purity (2) of extracted crude pigment at food grade level made it a suitable starting material for purification. Due to this, the purification methodology involving ammonium sulphate precipitation and dialysis resulted in achieving analytical grade purity of C-PC in a lesser number of steps. Characterization of purified C-phycocyanin demonstrates the spectral properties and compares its structural similarities to commercial analytical grade C-phycocyanin. Bioactivity evaluation of purified C-phycocyanin shows its potential as anti-oxidant compound with an IC50 value of 123.62 µg mL−1, and as anti-cancer compound with an IC50 value of 232.2 ± 4.2 µg. mL−1 with significant effects on cell migration and cell cycle arrest in G2 phase in HeLa cells.

Potential use of beneficial fungal microorganisms and C-phycocyanin extract for enhancing seed germination, seedling growth and biochemical traits of Solanum lycopersicum L.

BackgroundBiopriming as a new technique of seed treatment involves the application of beneficial microorganisms on the seed surface to stimulate seed germination, plant growth, and protect the seed from soil and seed-borne pathogens. The present investigation was carried out on seed germination, seedling vigor and biochemical traits of one of the most important vegetable crops (Tomato, Solanum lycopersicum L.). The treatments comprised viz. T1: Non primed seeds (Control), T2: Hydropriming, T3: Biopriming with C-phycocyanin (C-PC) (Spirulina platensis extract), T4: Biopriming with Trichoderma asperellum, T5: Biopriming with T. viride, T6: Biopriming with Beauveria bassiana.ResultsExtraction and purification of C-phycocyanin (C-PC) from the dry S. platensis powder using various methods was performed. The purity after dialyses was 0.49 and its ultimate purity (A620/A280) after ion-exchange chromatography was 4.64. The results on tomato seedlings revealed that the maximum germination percentage (100%), germination index (15.46 and 15.12), seedling length (10.67 cm), seedling dry weight (1.73 and 1.97 mg) and seedling length vigor index (1066.7) were recorded for tomato biopriming with T. viride, and B. bassiana (T5 and T6). Moreover, the quantitative estimation of total carbohydrates and total free amino acids contents in bioprimed tomato seedlings indicated a significantly higher amount with T. viride, followed by those bioprimed with T. asperellum, B. bassiana and C-PC extract.ConclusionThus, our results indicated that biopriming of tomato seeds with beneficial fungal inoculants and C-PC was very effective. The most operative biostimulants were those bioprimed with T. viride and B. bassiana compared to other biostimulants (T. asperellum and C-PC). Therefore, to ensure sustainable agriculture, this study offers new possibilities for the biopriming application as an alternative and ecological management strategy to chemical treatment and provides a valuable basis for improving seed germination.

Design strategies for C-phycocyanin purification: Process influence on purity grade

The performance of four purification designs combining fractional precipitation, ultrafiltration, and ion exchange chromatography (IEC) was compared in the purification of C-phycocyanin (C-PC) from the wet biomass of Spirulina platensis LEB-52. The purification strategies were evaluated in terms of purity grade, purification factor and recovery of the product. Additionally, the antioxidant activities of the clarified and purified C-PC extracts were compared. Initially, three polyethersulfone ultrafiltration membranes of different molecular weight cut-offs (30, 50, and 100 kDa) were evaluated in the purification of C-PC. The 30 and 50 kDa membranes showed statistically equal purification results (p > 0.05), and the 50 kDa membrane was selected to be used in the next steps. Then, different purification designs, which included fractional precipitation, ultrafiltration and IEC were applied. The purification design consisting of ultrafiltration followed by IEC with pH gradient elution was considered the most advantageous because it resulted in C-PC with a purity of 3.9, purification factor of 4.9-fold, and 79.7% recovery. In addition, the antioxidant capacity results showed that the extract with the highest purity (3.9) presented higher scavenging capacity against ABTS●+ (161.66 µmolTE.g−1) and peroxyl (1211.41 µmolTE.g−1) radicals when compared to the clarified extract (8.85 and 29.80 µmolTE.g−1, respectively). This work demonstrates the advantages of applying a purification design to C-PC, as it results in a product of high purification factor, recovery, and antioxidant activity, important characteristics due to the high added-value and biotechnological importance of this biomolecule.

Methods for Extraction, Isolation and Purification of C-phycocyanin: 50 years of research in review

Methods for Extraction, Isolation and Purification of C-phycocyanin: 50 years of research in review

Light-Harvesting Proteins and Biofilms on Iron Oxide Photoelectrodes

The so-called artificial photosynthesis has developed in at least two branches: semiconductor photoelectrochemistry with photoelectrochemical cells, and bio-molecular systems. Here we show bio-hybrid systems where metal oxide semiconductor photoelectrodes are fucntionalized at their surface with light harvesting proteins, specifically phycocyanin light antenna from blue-green algae. It has turned out in the early beginning of our studies that such funtionalization can yield a higher photocurrent and a higher hydrogen output. The actual underlying physico-chemical processes are not yet so well explored. We have therefore conducted electrochemical impedance studies on bio-hybride electrodes under physiological conditions such as to determine charge transfer processes. Moreover have we conducted x-ray photoemission studies on model systems in order to understand the valence band struc ture of the bio-electric interface and its charge transfer properties, particularly under dark conditions. The first bio-hybrid electrodes were used by Melvin Calvin in the late 1950s where he sublimated chlorophyll on aquadag graphite interdigital electrodes. This was done merely to study the bio-organic material. Helmut Tributsch' dye-sensitized solar cells (DSSC) in the early 1970s were ZnO electrodes, i.e. semiconductors further functionalized with natural absorbing dye molecules. Since, DSSCs have developed as a field in photovoltaic solar energy conversion technology. Meanwhile, photoelectrodes for solar water splitting in photoelectrochemical cells (PEC) evolved as an independent field. This field has recently emerged again and is virtually taking center stage. Also here, functionalization of traditional semicondcutor electrodes with bio-organic motifs is gaining more and more interest. Hydrogenase is a frequently used protein for the promotion of hydrogen evolution on photocathodes. We have recently coated the light harvesting antenna protein C-phycocyanin on iron oxide photoelectrodes. This approach was quite promising. We measured an increrased photocurrent density at the photoanode and an increased hydrogen evolution at the counter electrode. During our studies we were pointed to the question of charge transfer across the bio-electronic interface which is formed by biomolecules and metal oxides. Because the expressioan and purification of C-phycocyanin, which is extracted from cyanobacteria, is a costly and laborious process, we explored also the use of complete cyanobacteria on the photoelectrodes. The thylakoid membrane and cell membrane may pose electrical barriers between the biocatalytic or light harvesting components in the photosynthetic apparatus and the semicondcutor electrode underneath. It is therefore necessary to quantify this barrier. For this, we are performing electroanalytical experiments and x-ray spectroscopy experiments operando and in situ, in the latter case with an actual anabaena biofilm with ambient pressure XPS on a bio-electrochemica cell.[1] J. Ihssen, A. Braun, G. Faccio, K. Gajda-Schrantz, L. Thöny-Meyer; Light harvesting proteins for solar fuel generation in bioengineered photoelectrochemical cells; Current Protein and Peptide Science, 2014, 15 (4),374-384.[2] D. K. Bora, Y. Hu, S. Thiess, S. Erat, X. Feng, S. Mukherjee, G. Fortunato, N. Gaillard, R. Toth, K. Gajda-Schrantz, W. Drube, M. Grätzel, J. Guo, J. Zhu, E.C. Constable, D.D. Sarma, H. Wang, A. Braun, Between Photocatalysis and Photosynthesis: Synchrotron spectroscopy methods on molecules and materials for solar hydrogen generation, J. Electron Spectr. Rel. Phenom. 2013, 190 A, 93-105.[3] D.K. Bora, A.A. Rozhkova, K. Schrantz, P.P. Wyss, A. Braun, T. Graule, E.C. Constable, Functionalization of Nanostructured Hematite Thin-Film Electrodes with the Light-Harvesting Membrane Protein C-Phycocyanin Yields an Enhanced Photocurrent, Advanced Functional Materials 2012, 22 (3) 490-502.[4] D.K. Bora, A. Braun, E.C. Constable, “In rust we trust”. Hematite the prospective inorganic backbone for artificial photosynthesis, Energy Environ. Sci., 2013,6, 407-425.[5] A Braun, F. Boudoire, D. K. Bora, G. Faccio, Y. Hu, A. Kroll, B. S. Mun, S. T. Wilson, Biological components and bio-electronic interfaces of water splitting photo-electrodes for solar hydrogen production, Chem. Eur. J. 2015, 21(11), 4188-4199.

Purification of C-phycocyanin from Spirulina platensis in aqueous two-phase systems using an experimental design

C-phycocyanin from Spirulina platensis was purified in aqueous two-phase systems (ATPS) of polyethylene glycol (PEG)/potassium phosphate, varying the molar mass of the PEG. Results using a full factorial design showed that an increase in the concentration of salt and decrease in the concentration of PEG caused an increment in the purification factor for all the ATPS studied. Optimization of the conditions of the purification was studied using a central composite rotatable design for each molar mass of PEG. The ATPS composed of 7% (w/w) PEG 1500 or 4% (w/w) PEG 8000 (g/gmol) and 23 or 22.5% (w/w) of phosphate resulted a purification factor of 1.6-fold for C-phycocyanin, with total and 57% recovery, respectively. Process conditions were optimized for the purification factor for the system with PEG 1500. The ATPS with 4% (w/w) PEG 4000 or 4% (w/w) PEG 6000 and 21% (w/w) phosphate resulted purification factors of 2.1 and 2.2-fold, recovering 100% and 73.5%, respectively of C-phycocyanin in the top phase.

Extraction and Purification of C-Phycocyanin from Spirulina platensis Using Aqueous Two Phase Extraction and its Applications

Extraction and Purification of C-Phycocyanin from Spirulina platensis Using Aqueous Two Phase Extraction and its Applications

C-Phycocyanin from Oscillatoria tenuis exhibited an antioxidant and in vitro antiproliferative activity through induction of apoptosis and G0/G1 cell cycle arrest

This study was undertaken to develop an efficient single step chromatographic method for purification of C-phycocyanin (CPC) from species of Oscillatoria tenuis. Purification of CPC involves a multistep treatment of the crude extract by precipitation with ammonium sulphate, followed by gel filtration chromatography. Pure CPC was finally obtained from O. tenuis with purity ratio (A620/A280) 4.88. SDS–PAGE of pure CPC yielded two bands corresponding to α and β subunits; the molecular weight of α subunit is 17.0kDa, whereas the molecular weight of β subunit is 19.5kDa. Fluorescence and phase contrast microscopy revealed characteristic apoptotic features like cell shrinkage, membrane blebbing, nuclear condensation and DNA fragmentation. CPC exhibited antioxidant and antiproliferative activity against human cancer cells through apoptosis; nuclear apoptosis induction was accompanied by G0/G1 phase arrest and DNA fragmentation. CPC is a natural pigment with potential as an anticancer agent.

Purification of C-Phycocyanin from Spirulina platensis by Single-Step Ion-Exchange Chromatography

C-Phycocyanin is a natural blue pigment with many commercial applications in foods, cosmetics, and medicines. In this paper we describe the extraction and purification of C-phycocyanin from the cyanobacterium Spirulina platensis. The procedure is based on adsorption of impurities with chitosan and activated charcoal then one-step ion-exchange chromatography. The dry algal powder was soaked in potassium phosphate buffer for 2 h to furnish crude phycocyanin extract of purity 0.93. The crude extract was then treated with chitosan and activated charcoal for 5 min, which increased the purity to 2.78. After chromatography on DEAE Sephadex A-25, the purity of phycocyanin was improved to 4.3. The identity of the purified phycocyanin was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoretic analysis and by spectroscopic measurements (UV–visible spectrophotometry and spectrofluorimetry). Compared with conventional methods, this method was simple, inexpensive, and time-saving.

Extraction and purification of C-phycocyanin from Spirulina platensis in conventional and integrated aqueous two-phase systems

Crude C-phycocyanin extracts from Spirulina platensis, with and without cell debris, were purified in aqueous two-phase systems ( ATPS) varying the molar mass of the PEG, at pH 6, more promising than at pH 7 . For the assays carried out with each molar mass, with distinct percentages of PEG and salt, the concentrations (mg mL-1), purities (OD 620/OD 280) and C-phycocyanin partition coefficients of each system were determined and compared. The process for the primary purification of intracellular C-phycocyanin with cell debris (integrated), composed of 15% of PEG 1500 and 13% of potassium phosphate and 5% of PEG 4000 and 18% of salt, produced C-phycocyanin concentrations of, respectively, 1.60 and 2.67 mg mL-1 in the top phase. The purities were of 0.73 and 0.79 respectively, values higher than those obtained in the systems without cells.

Extraction and Purification of C-Phycocyanin from Spirulina Platensis Employing Aqueous Two Phase Systems

Aqueous two phase extraction was employed directly to the cell homogenate of Spirulina platensis for the downstream processing of C-phycocyanin. This enables integrating the process steps of cell removal, extraction and concentration into a single unit operation of aqueous two phase extraction. The effect of different parameters such as molecular weight, tie line length, volume ratio and neutral salt (NaCl) were studied employing PEG 4000/potassium phosphate system and PEG 1000/sodium sulfate system to select the best system among the two. For PEG 4000/potassium phosphate system at 13.12% TLL, and volume ratio 0.45, 90.34% yield of phycocyanin with purity of 0.73 was obtained. In the case of PEG 1000 /Na2SO4 system at 34.98 % TLL and volume ratio of 1, 97.62 % yield of phycocyanin with a purity of 0.41 was achieved. Effect of NaCl concentration shows a significant decrease in K of phycocyanin in PEG 1000/Na2SO4 system as compared to PEG 4000/potassium phosphate system. PEG 4000 and potassium phosphate system was found to be better than PEG 1000 and sodium sulfate system for the extraction and purification of C-phycocyanin.

Improved procedure for separation and purification of Arthronema africanum phycobiliproteins

A rapid, inexpensive and reliable procedure for separation and purification of C-phycocyanin (C-PC) and allophycocyanin (APC) from Arthronema africanum based on a previously described rivanol-sulfate method for C-PC purification was developed. Exclusion of NaCl from the extraction buffer resulted in complete separation of APC and C-PC, two-fold reduction of rivanol treatments, and a higher yield and purity of C-PC. Pure C-PC (A(620)/A(280) of 4.52) and APC (A(652)/A(280) of 2.41) were obtained. The estimated molecular masses of the alpha and beta subunits were 17 and 19 kDsmall a, Cyrillic for capital ES, Cyrillic-phycocyanin and 16 and 18 kDsmall a, Cyrillic for APC, respectively. The overall C-PC recovery of 55% (w/w) from its content (100 mg) in the crude extract was 10-20% higher than so far reported. The procedure appears promising for scaling up and broader applications.

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.

Purification of c-phycocyanin from Spirulina fusiformis and its effect on the induction of urokinase-type plasminogen activator from calf pulmonary endothelial cells

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated. The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method. The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.

A simple method for efficient separation and purification of c-phycocyanin and allophycocyanin from Spirulina platensis

c-Phycocyanin and allophycocyanin were separated and purified from Spirulina platensis by precipitation with ammonium sulphate, ion exchange chromatography and gel filtration chromatography. Pure c-phycocyanin and allophycocyanin were finally obtained with an A620/A280value of 5.06 and an A655/A280 value of 5.34, respectively.