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Cyanobacterial phycobiliproteins, such as phycoerythrin (PE) and phycocyanin (PC), are colored potential bioactive proteins that have antioxidant and antimicrobial properties. In this study, we formulated a new food prototype based on PE and PC-fortified low-fat yogurt and cream cheese. Four distinct low-fat yogurt and cream cheese products were manufactured, including a control group (No PE and PC), samples produced with phycoerythrin (+ PE), samples produced with phycocyanin (+ PC), and samples produced with both phycoerythrin and phycocyanin (PC + PE). Afterwards statistically compared the physicochemical composition, colorimetric properties, antioxidant and antimicrobial activities, and sensory profile of the fortified foods at 4 °C and 8 °C for 28 and 42 days. Additionally, we confirmed that PE and PC are not toxic to Caenorhabditis elegans at concentrations up to 1 mg/mL. The results showed that the MIC of PE and PC against E. coli was significantly higher than against S. aureus (3.12 ± 0.05 µg/mL vs. 1.56 ± 0.01 µg/mL, respectively; p ≤ 0.05). Additionally, the maximum diameter of the inhibition zone of PE and PC against S. aureus was significantly higher than against E. coli (6.6 ± 0.011 mm vs. 11.66 ± 0.02 mm, respectively; p ≤ 0.05). Results of color parameters showed that the control group had significantly higher L* values than the samples enriched with PE and PC. Moreover PE and PC significantly increased the a* and b* values respectively. The amount of ΔE in the control yogurts and cream cheese was higher than in the samples with PE and PC. Overall, the results showed that adding PE and PC had a significant effect on all measured factors (p < 0.01). Cream cheeses and low-fat yogurts enriched with either PE or PE + PC had the greatest antioxidant activity and the lowest number of psychrophilic bacteria and mold, and yeast counts at the end of the test period. Therefore, low-fat yogurt and cream cheese containing cyanobacterial PE and PC can be considered an innovative dairy product for the food industry. This study marks the initial effort to employ PE and PC derived from Nostoc sp. and Spirulina sp. as antioxidant and antimicrobial agents in the food industry., Competing Interests: Competing interests The authors declare no competing interests. Ethical statement All methods were carried out following relevant guidelines and regulations. The authors did not do any experiments on humans and/or the use of human tissue samples. All experimental protocols and panelists involved in the study were approved by the ethics committee of Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Participants gave their consent to take part and use their information. The full name of the ethics committee that approved the study is Dr Fahimeh Nemati and Dr Sarvenaz Falsafi., (© 2024. The Author(s).)

Phycobilisomes (PBSs) are light-harvesting antenna complexes in cyanobacteria that adapt to diverse light environments through the use of phycobiliproteins within the PBS structures. Freshwater cyanobacteria, such as Synechococcus elongatus PCC 7942, thrive under red light because of the presence of phycocyanin (PC) and its chromophore, phycocyanobilin (PCB), in the PBS. Cyanobacteria in shorter-wavelength light environments such as green light, employ phycoerythrin paired with phycoerythrobilin (PEB) along with PC in the PBS. Synthetic biology studies have shown that PEB production can be achieved by expression of the heterologous PEB synthases 15,16-dihydrobiliverdin:ferredoxin oxidoreductase (PebA) and PEB:ferredoxin oxidoreductase (PebB), leading to PEB accumulation and cellular browning. This approach is genetically unstable, and the properties of the resulting PEB-bound PBS complexes remain uncharacterized. In this study, we engineered a novel strain of Synechococcus 7942 PEB1 with finely tuned control of PEB biosynthesis. PEB1 exhibited a reversible change in the color of the culture from green to brown and pink based on PebA and PebB induction levels. High induction led to complete PCB-to-PEB substitution, causing the disassembly of the PBS rod complex. In contrast, low induction levels of PebA and PebB resulted in the formation of a stable chimeric PBS complex with partial PCB-to-PEB substitution. This acclimation enabled efficient light harvesting in the green spectrum and energy transfer to the photosynthetic reaction center. These findings, which improve our understanding of PBS and highlight the structural importance of the bilin composition, provide a foundation for future studies on PBS adaptation in bioengineering, synthetic biology, and renewable energy.

Cluster 5 picocyanobacteria significantly contribute to primary productivity in aquatic ecosystems. Estuarine populations are highly diverse and consist of many co-occurring strains, but their physiology remains largely understudied. In this study, we characterized 17 novel estuarine picocyanobacterial strains. Phylogenetic analysis of the 16S rRNA and pigment genes (cpcB and cpeBA) uncovered multiple estuarine and freshwater-related clusters and pigment types. Assays with five representative strains (three phycocyanin rich and two phycoerythrin rich) under temperature (10-30°C), light (10-190 μmol photons m -2  s -1 ), and salinity (2-14 PSU) gradients revealed distinct growth optima and tolerance, indicating that genetic variability was accompanied by physiological diversity. Adaptability to environmental conditions was associated with differential pigment content and photosynthetic performance. Amplicon sequence variants at a coastal and an offshore station linked population dynamics with phylogenetic clusters, supporting that strains isolated in this study represent key ecotypes within the Baltic Sea picocyanobacterial community. The functional diversity found within strains with the same pigment type suggests that understanding estuarine picocyanobacterial ecology requires analysis beyond the phycocyanin and phycoerythrin divide. This new knowledge of the environmental preferences in estuarine picocyanobacteria is important for understanding and evaluating productivity in current and future ecosystems., (© 2023 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Marine Synechococcus cyanobacteria owe their ubiquity in part to the wide pigment diversity of their light-harvesting complexes. In open ocean waters, cells predominantly possess sophisticated antennae with rods composed of phycocyanin and two types of phycoerythrins (PEI and PEII). Some strains are specialized for harvesting either green or blue light, while others can dynamically modify their light absorption spectrum to match the dominant ambient color. This process, called type IV chromatic acclimation (CA4), has been linked to the presence of a small genomic island occurring in two configurations (CA4-A and CA4-B). While the CA4-A process has been partially characterized, the CA4-B process has remained an enigma. Here we characterize the function of two members of the phycobilin lyase E/F clan, MpeW and MpeQ, in Synechococcus sp. strain A15-62 and demonstrate their critical role in CA4-B. While MpeW, encoded in the CA4-B island and up-regulated in green light, attaches the green light-absorbing chromophore phycoerythrobilin to cysteine-83 of the PEII α-subunit in green light, MpeQ binds phycoerythrobilin and isomerizes it into the blue light-absorbing phycourobilin at the same site in blue light, reversing the relationship of MpeZ and MpeY in the CA4-A strain RS9916. Our data thus reveal key molecular differences between the two types of chromatic acclimaters, both highly abundant but occupying distinct complementary ecological niches in the ocean. They also support an evolutionary scenario whereby CA4-B island acquisition allowed former blue light specialists to become chromatic acclimaters, while former green light specialists would have acquired this capacity by gaining a CA4-A island., Competing Interests: The authors declare no competing interest.

Background: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry., Methods: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<>) were used., Results: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes., Conclusions: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes., (Copyright 2002 Wiley-Liss, Inc.)

Background: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product., Methods: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC., Results: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%., Conclusion: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.

The supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin-HLA-DR, Texas red-CD34, allophycocyanin-CD15, and R123 were fractionated using four-color immunofluorescence cell sorting. Cells expressing CD34 but not HLA-DR and CD15 (CD34+ HLA-DR- CD15-) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R+) or cycling, R123 bright (R++) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34+ HLA-DR- CD15- R+ cells contained larger numbers of blast cells and colony forming units than CD34+ HLA-DR- CD15- R++ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.

Single crystal x-ray diffraction studies are proceeding on two phycobiliproteins: C-Phycocyanin from Anabaena variabilis and B-Phycoerythrin from Porphyridium cruentum. C-Phycocyanin consists of six alpha and six beta subunits. A three-dimensional x-ray diffraction study of this light-harvesting protein (P6(3); a = b = 154.1 A, c = 40.1 A) at 5-A resolution shows that the molecule is 110 A in diameter, 40 A thick, with a 20-A diameter central channel. It can be divided into three separate domains related by the crystallographic 3 and contains long, columnar regions of density, presumed to represent alpha helix. B-Phycoerythrin is composed of 6 alpha, 6 beta, and a gamma subunit. A 5.25-A resolution study of this protein (R3, a = b = 189 A, c = 60 A) indicates that the molecule is 107 A in diameter and 55 A thick, has 32-D3 symmetry, and can be divided into six regions. The gamma chain lies on a crystallographic 3 and undergoes symmetric disordering. A region of low and unstructured density in the center of the molecule is presumably occupied by this gamma chains. These two low resolution structures are compared with regard to differences in quaternary structure.

Previous spectroscopic studies on the phycocyanobilin-containing peptide beta-2T from Synechococcus sp. 6301 C-phycocyanin and the phycoerythrobilin-containing peptide beta-2TP from Porphyridium cruentum B-phycoerythrin indicated a different single thioether mode of attachment, postulated to be through the D-ring of the tetrapyrrole, in contrast to the A-ring linkage established for the other singly linked bilins in these proteins (Bishop, J.E., Lagarias, J.C., Nagy, J. O., Schoenleber, R.W., Rapoport, H., Klotz, A.V., and Glazer, A.N. (1986) J. Biol. Chem. 261, 6790-6796; Klotz, A.V., Glazer, A.N., Bishop, J.E., Nagy, J.O., and Rapoport, H. (1986) J. Biol. Chem. 261, 6797-6805). The crystal structure of Agmenellum quadruplicatum C-phycocyanin at 2.5-A resolution (Schirmer, T., Bode, W., and Huber, R. (1987) J. Mol. Biol., 196, 677-695) supports an A-ring linkage for all three phycocyanobilins. Consequently we have re-evaluated our proposed structural assignments by further 1H NMR studies. Two-dimensional homonuclear correlated and nuclear Overhauser enhancement spectroscopic data presented here show that all three bilins in Synechococcus 6301 C-phycocyanin are attached solely through the A-ring, complementary to the crystallographic data. The evidence from the NMR data for all bilin peptides examined includes the dipoledipole interactions of the 5-H with the 3-H, 3'-H, and a pyrrole methyl group (7-CH3); the corresponding interactions would not be possible in a D-ring-linked bilin. The 5-H also consistently exhibits allylic J-coupling to the 3-H, supporting A-ring linkage assignment. These data are inconsistent with the alternative D-ring linkage assignment since this would involve J-coupling through five bonds. Examination of the phycoerythrobilin beta-2 position in B-phycoerythrin also reveals an A-ring type of attachment by similar criteria. We conclude that all singly linked bilins are attached through the A-ring.

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.

Phycoerythrobilin and phycocyanobilin are covalently attached to the apoproteins of phycoerythrins and phycocyanins. One linkage consists of an ester bond between the hydroxy group of a serine residue and the propionate side chain on one of the inner pyrrole rings (probably ring C). The other linkage is a labile thioether bond between a cysteine residue and the two-carbon side chain on pyrrole ring A. This side chain and both of the alpha-positions of the ring A are in the reduced state. This constitutes an important structural revision, since, in the structures currently accepted for the phycobilins, the two-carbon side chain on ring A is depicted as an ethylidene grouping and this has been regarded not only as a very characteristic feature of the phycobilins, but also as a probable structural feature of the chromophore of phytochrome, largely on the basis of other analogies with the phycobilins. The ethylidene-containing structures apply instead to artefact forms of the pigments released from the apoproteins by treatment with hot methanol. Cleavage of the ring-A linkage involves an elimination reaction releasing the cysteine residue and generating a double bond in the ring-A side chain. During cleavage in methanol the direction of the elimination is towards the ring, generating the ethylidene double bond. Since this is linked to the conjugated system, the methanol-released pigments differ spectrally from the native phycobilins. During acid-catalysed release of the pigments, the elimination apparently goes in the opposite direction, generating a double bond at the outer position of the side chain. Since this double bond is not linked to the conjugated system, the acid-released pigments remain spectrally identical with their protein-bound counterparts.

The energies of the highest occupied and lowest empty molecular orbitals were calculated for the chromophore groups of the proteins phycocyanin and phycoerythrin. These tetrapyrrole groups on the algal proteins are shown to provide them with the potential of ating as efficient electron donors and acceptors. In addition, the pi electron charges and bond orders were also computed.

Addition of phycoerythrobilin (PEB) to apophycocyanin at pH 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues alpha-Cys-84 and beta-Cys-82 to be the sites of bilin addition. The product of PEB addition at the alpha-Cys-84 site was shown by 1H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the beta-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a lambda max of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins.

Single crystal x-ray diffraction investigations are in progress on two phycobiliproteins. C-phycocyanin from Anabaena variabilis crystallizes in space group P63 with a = b = 154 A and c = 40 A. The crystallographic asymmetric unit is (alphabeta)2 with a total molecular mass of 7.0-10(4) daltons. B-phycoerythrin from Porphyridium cruentum crystallizes in space group R3 with a = b = 189 A and c = 60 A. This molecule has the unusual molecular stoichiometry (alphabeta)6gamma and the crystallographic asymmetric unit is (alphabeta)2gamma1/3. This requires that the gamma chain undergo a perfect 3-fold disordering about the crystallographic 3 axis, i.e. the gamma chain must occupy three symmetry-equivalent positions, each with an occupancy of one-third.

1. R-phycoerythrin from Ceramium rubrum and C-phycocyanin from Nostoc muscorum have been obtained in purified form by fractional crystallization, followed by chromatography and gel-filtration. 2. The amino acid composition of both chromoproteins was determined; 85% of the total weight of both was recovered as amino acids. 3. Alanine was identified as the only C-terminal amino acid of R-phycoerythrin, each molecule of which contained about 12 terminal groups. 4. Serine was identified as the only C-terminal group of C-phycocyanin.

The phycobilin pigments were freshly extracted from Porphyra naiadum in the cold. At least two types of phycoerythrin (I and II) can be distinguished by electrophoresis, chromatography, and spectral characteristics. At pH 5.0 phycoerythrin II has a relatively large negative charge, while phycoerythrin I is nearly iso-electric. At pH 7.0, however, phycoerythrin I has the larger negative charge. Mobilities have been calculated by visual measurement of electrophoresis. Phycoerythrin II can be converted to phycoerythrin I by storing at pH 7.0. Chromatography indicates at least two types of phycocyanin as well.

1. R-, B- and C-phycoerythrins and R- and C-phycocyanins were isolated and purified on a preparative scale by calcium phosphate chromatography, ammonium sulphate fractionation and crystallization. 2. The N-terminal residues of these biliproteins were analysed. Methionine is the only N-terminal residue of all the phycoerythrins, there being about 14 N-terminal residues per molecule of R- and B-phycoerythrins (mol.wt. 290000) and about 8 per molecule of C-phycoerythrin (mol.wt. 226000). Threonine (1 residue) is N-terminal in C-phycocyanin (mol.wt. 138000), and both threonine (about 1.3 residues) and methionine (5 residues) are N-terminal in R-phycocyanin (mol.wt. 273000). 3. Results suggest that the apoproteins of the various phycoerythrins are closely related, whereas C-phycocyanin has quite a different gross structure, and that R-phycocyanin contains two types of sub-unit, one related to C-phycocyanin and the other to the phycoerythrins.

1. The fluorescence spectra of the alga Porphyridium have been recorded as energy distribution curves for eleven different incident wave lengths of monochromatic incident light between wave lengths 405 and 546 mmicro. 2. In these spectra chlorophyll fluorescence predominates when the incident light is in the blue part of the spectrum which is strongly absorbed by chlorophyll. 3. For blue-green and green light the spectrum excited in Porphyridium contains in addition to chlorophyll fluorescence, the fluorescence bands characteristic of phycoerythrin and of phycocyanin. 4. From these spectra the approximate curves for the fluorescence of the individual pigments phycoerythrin, phycocyanin, and chlorophyll in the living material have been derived and the relative intensity of each of them has been obtained for each of the eleven incident wave lengths. 5. The effectiveness spectrum for the excitation of the fluorescence of these three pigments in vivo has been plotted. 6. From comparisons of the effectiveness spectrum for the excitation of each of these pigments it appears that both phycocyanin and chlorophyll receive energy from light which is absorbed by phycoerythrin. 7. It is suggested that phycocyanin may be an intermediate in the resonance transfer of energy from phycoerythrin to chlorophyll. 8. Since phycoerythrin and phycocyanin transfer energy to chlorophyll, it appears probable that chlorophyll plays a specific chemical role in photosynthesis in addition to acting as a light absorber.

Background: The use of natural ingredients in skincare products has become a topic of interest in contemporary society. The adverse effects and environmental risks of synthetic compounds have prompted studies on the use of photosynthetic organisms as sustainable and ecofriendly sources of effective ingredients. Natural extracts have gained attention in the cosmetic industry, especially those derived from plants, algae, and cyanobacteria. Cyanobacteria have gained prominence in the cosmetic industry because of their low culture requirements, rapid growth rates, and capacity to produce different bioactive metabolites. As a result, cyanobacteria are economically viable and sustainable resources. Objective: We aimed to determine the effect of coating with natural phycocyanin and phycoerythrin pigments from two cyanobacterial species, Spirulina platensis and Nostoc sp., on the properties of three cosmetic products: soap, anti-acne face wash, and hand sanitizer gel. Methods: After cyanobacterial culture, the pigments were extracted, purified, and coated with the stabilizer, chitosan. Thereafter, cosmetic products were formulated with phycocyanin and phycoerythrin, and their viscosity, pH, stability, and antioxidant and antibacterial properties against bacteria were evaluated using the appropriate assays. Results: The anti-acne wash gel and soap had better properties when coated with both pigments and phycocyanin alone, whereas the hand sanitizer gel had better properties when coated with phycoerythrin alone and both pigments. Conclusion: Overall, three coated and stable cosmetic products can be produced using cyanobacterial pigments owing to their high fortifying capacity.

Abstract Cyanobacterial phycocyanin and phycoerythrin are gaining commercial interest due to their nutrition and healthcare values. This research analyzed the biomass accumulation and pigment production of two strains of Leptolyngbya under different combinations of light colors and intensities. The results showed that while Leptolyngbya sp.4 B1 (B1) produced all phycobiliproteins, Leptolyngbya sp.5 F2 (F2) only had phycocyanin and allophycocyanin. Both the color of the light and its light intensity affect the biomass accumulation and phycoerythrin concentration in strain B1. Although white light at medium intensity (50 μmol m-2 s-1) causes greater biomass accumulation (1.66 ± 0.13 gDW L-1), low-intensity (25 μmol m-2 s-1) green light induces lower biomass accumulation with twice the pigment content (87.70 ± 2.46 mg gDW -1), culminating in 71% greater productivity. In contrast, for the F2 strain, light intensity positively influenced biomass and pigment accumulation, being observed 2.25 ± 0.10 gDW L-1 under white light at 100 μmol m-2 s-1 and higher phycocyanin concentration (138.38 ± 3.46 mg gDW -1) under red light at 100 μmol m-2 s-1. These findings provide insights into optimizing the growth conditions by altering the intensity and wavelength of light for future production of phycocyanin and phycoerythrin from local cyanobacteria.

Suitability of microalgae valorization mainly depends on its biochemical composition. Overall, among all microalgal derivatives, pigments currently stand out as the major added-value component. While it is well recognized that microalgal growth conditions strongly affect biomass composition, final tuning of already grown microalgae has been scarcely studied. Herein, pigment crude extract and debris biomass composition of an already grown microalgal consortium was evaluated after a short-term exposure (90 min) to different levels of irradiance (15, 50, 120 μmol m−2 s−1) and sulfide concentrations (0, 3.2, 16 mg L−1). Although lipid, protein, and carbohydrate contents of debris biomass were not decisively modified by the short-term exposures, pigments content of the crude extracts were strongly modified after 90-min exposure at given sulfide and irradiance conditions. Particularly, a higher content of chlorophyll a, chlorophyll b, and total carotenoids was estimated at an optimal sulfide concentration of 5 mg L−1, and the higher irradiance of 120 μmol m−2 s−1. Contrarily, the average irradiation level of 50 μmol m−2 s−1 and the absence of sulfide stimulated the production of phycoerythrin and phycocyanin which could be increased by 65 and 50%, respectively. Thus, a final qualitative and quantitative tuning of pigment content is plainly achievable on grown microalgal biomass, in a reduced exposure time, at given irradiance or sulfide conditions. [ABSTRACT FROM AUTHOR]

The research in the field of food storage and preservation intensified in recent years to develop alternative preservatives and colorants to chemical ones. The algal preservatives and colorants have emerged as a viable green alternative in this context. The primary food preservatives from the algae are phycobiliproteins (PBPs) and their primary constituents: allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE). As the foods have been subjected to the adverse environmental conditions during the production, storage, and utilization stages, it is important to research the determinants of the thermal, acidic, light, and color stability of the PBPs in these adverse environmental conditions such as light and acid exposure as well as elevated and sub-zero temperatures relevant to the food storage. Although there have been around 60 papers published in this research field, there has been no review of the research on the stability of the PBPs in these adverse environmental conditions relevant to the food storage. Hence, this paper reviews the research in this field as a first-ever population study. These studies reveal that these PBPs are stable at a wide range of acid and light exposure, and temperatures, making them perfect for the foods and beverages as a green alternative to chemical preservatives. Furthermore, a wide range of preservatives improve the stability of these PBPs through their close interaction with the PBPs and encapsulation of the PBPs is an efficient way to improve their stability, potentially using the nanotechnology. A couple of recommendations were made for the further research. [ABSTRACT FROM AUTHOR]

[Display omitted] • P. purpureum and P. aerugineum are rich in PE and PC, respectively. • Both Porphyridium species exhibited optimal growth under white light. • Blue light enhanced both the content and proportion of core phycobiliproteins. • The highest PE content in P. purpureum reached 69.37 ± 0.92 mg/g DW under blue light. • P. aerugineum achieved the highest PC titer of 216.92 ± 1.90 mg/L under white light. Microalgae not only fix carbon dioxide, but also represent a promising alternative resource for the production of proteins, lipids, and polysaccharides. This study employed two Porphyridium strains to compare their responses under different light qualities. P. purpureum up-regulated the content (up to 69.37 ± 0.92 mg/g DW) and proportion of phycoerythrin to enhance light absorption, which led to the accumulation of total soluble proteins, neutral lipids and exopolysaccharides under blue light. In contrast, P. aerugineum primarily improved the light energy utilization by increasing phycocyanin levels (up to 81.10 ± 0.60 mg/g DW), resulting in the degradation of neutral lipids and the accumulation of exopolysaccharides. Given the biomass, the highest yields of phycoerythrin (169.61 ± 2.90 mg/L) and phycocyanin (216.92 ± 1.90 mg/L) were achieved by P. purpureum and P. aerugineum cultured under white light, respectively. These findings indicate that Porphyridium can serve as a valuable resource for phycobiliprotein production, with biomolecules synthesis being tightly regulated by light quality. [ABSTRACT FROM AUTHOR]

Phycocyanin (PC) and phycoerythrin (PE) stand out among the phycobiliproteins (PCBs) known to date. These bioactive protein complexes found in certain organisms, particularly cyanobacteria, red algae, and some cryptophytes have a wide range of applications from foods, cosmetics, and healthcare to biotechnology. Reviews focusing only on PC and PE are lacking. Therefore, in this review, an overview of PCBs was provided first. To ensure high extraction yields and purity, the optimization of PC and PE production was discussed followed by the extraction of PC and PE highlighting the novel techniques used. Multifarious applications and biofunctional uses, as well as commercialization of the PCBs in various forms, were thereafter described. Available reports show that culture conditions including light quality affect the production of PC and PE. In addition, the pigments have wide applications in the healthcare, food, biotech, and cosmetics industries. [Display omitted] • A structural overview of the phycobiliproteins in the phycobilisome was presented. • Optimization of phycocyanin (PC) and phycoerythrin (PE) production was discussed. • Pleiotropic applications and biofunctional uses of PC and PE were described. • Some commercial endeavors based on the phycobiliproteins were presented. [ABSTRACT FROM AUTHOR]

The present study evaluated the effect of multiple variables (drying time, drying temperature, biomass/solvent ratio, glass beads/biomass ratio, extraction time, and extraction speed) in the solubilization of three different phycobiliproteins (C-PC, APC, and PE) from a thermotolerant Oscillatoria sp. The strain was grown in BG11 media (28 °C, light: dark cycle of 12:12 h at 100 µmol·m−2·s−1, 20 days) and the experiments were conducted according to a two-level randomized factorial design with six center points (38 runs). Results show that biomass/solvent ratio, glass beads/biomass ratio, and extraction time, are the most significant variables in the extraction of all three proteins, whereas the glass beads/biomass ratio and extraction time significantly affect their purity. The optimized conditions allow a statistical increase in the concentration of C-PC, APC, and PE extracted from the biomass; however, the purity was lower in comparison with the expected value. The latter occurs due to a larger biomass/solvent ratio and longer extraction times, which enhanced the solubility of other hydrophilic metabolites (proteins and carbohydrates, etc.). [ABSTRACT FROM AUTHOR]

The loss of density and elasticity, the appearance of wrinkles and hyperpigmentation are among the first noticeable signs of skin aging. Beyond UV radiation and oxidative stress, matrix metalloproteinases (MMPs) assume a preponderant role in the process, since their deregulation results in the degradation of most extracellular matrix components. In this survey, four cyanobacteria strains were explored for their capacity to produce secondary metabolites with biotechnological potential for use in anti-aging formulations. Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 from freshwater ecosystems, and Leptolyngbya cf. ectocarpi LEGE 11479 and Nodosilinea nodulosa LEGE 06104 from marine habitats were sequentially extracted with acetone and water, and extracts were analyzed for their toxicity in cell lines with key roles in the skin context (HaCAT, 3T3L1, and hCMEC). The non-toxic extracts were chemically characterized in terms of proteins, carotenoids, phenols, and chlorophyll a, and their anti-aging potential was explored through their ability to scavenge the physiological free radical superoxide anion radical (O2•−), to reduce the activity of the MMPs elastase and hyaluronidase, to inhibit tyrosinase and thus avoid melanin production, and to block UV-B radiation (sun protection factor, SPF). Leptolyngbya species stood out for anti-aging purposes: L. boryana LEGE 15486 presented a remarkable SPF of 19 (at 200 µg/mL), being among the best species regarding O2•− scavenging, (IC50 = 99.50 µg/mL) and also being able to inhibit tyrosinase (IC25 = 784 µg/mL), proving to be promising against UV-induced skin-aging; L. ectocarpi LEGE 11479 was more efficient in inhibiting MMPs (hyaluronidase, IC50 = 863 µg/mL; elastase, IC50 = 391 µg/mL), thus being the choice to retard dermal density loss. Principal component analysis (PCA) of the data allowed the grouping of extracts into three groups, according to their chemical composition; the correlation of carotenoids and chlorophyll a with MMPs activity (p < 0.01), O2•− scavenging with phenolic compounds (p < 0.01), and phycocyanin and allophycocyanin with SPF, pointing to these compounds in particular as responsible for UV-B blockage. This original survey explores, for the first time, the biotechnological potential of these cyanobacteria strains in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms. [ABSTRACT FROM AUTHOR]

Featured Application: Hybrid pigments from synthetic or natural nanoclays confer organic compounds stabilization as the main advantage. Nanoclays adsorb microalgae pigments, which improves the optical, mechanical and thermal properties of adsorbed organic compounds and the material in applications. This work provides the stabilization of phycocyanin and phycoethrin proteins, which can be used in several applications including coatings, polymer additives (i.e., 3D printing), cosmetics, and textile industries. C-Phycocyanin (PC) and B-phycoerythrin (PE) are light-harvesting water-soluble phycobiliproteins from microalgae that belong mainly to the cyanobaceria and rhodhophytes families. Different methods have been developed for PC and PE extraction and purification from microalgae, and offer a high potential for their use as additives in sectors such as food and cosmetics. However, the main limitations of using these dyes are the sensitivity of their environmental factors, such as light fastness, temperature, and pH. We successfully employed safe lamellar nanoclays such as montmorillonite (M) and Laponite (L) for phycobiliproteins stabilization, as we did before with other natural dyes. We obtained a wide color gamut from blues to pink by combining four different factors under synthesis conditions: three dye concentrations; two laminar nanoclay sizes; a two nanoclay surface modifiers combination with cetylpyridinium bromide (CPB) and a coupling agent (3-Aminopropyl) triethoxysilane. The experimental conditions were defined according to a multilevel factorial design of experiment (DOE) to study the factors interacting in the final hybrid pigment characteristics. In both M and L, the d001 distance (nm) increased due to PC and PE adsorption. The best conditions to increase the basal space depend on the nanoclay structure, and it is better to use the surfactant for M, and silane modification for L. In addition, optical and thermal PE and PC properties significantly improved. We show the optimal synthesis conditions to increase PC and PE adsorption using the high dye concentration, with surfactant and silane depending on the nanoclay. The hybrid pigments from these phycobiliproteins offer the opportunity to perform several industrial applications, including in polymer additives, cosmetics, and packaging. [ABSTRACT FROM AUTHOR]

Picophytoplankton in the Baltic Sea includes the simplest unicellular cyanoprokaryotes (Synechococcus / Cyanobium) and photosynthetic picoeukaryotes (PPE). Picophytoplankton are thought to be a key component of the phytoplankton community, but their seasonal dynamics and relationships with nutrients and temperature are largely unknown. We monitored pico- and larger phytoplankton at a coastal site in Kalmar Sound (K-Station) weekly during 2018. Among the cyanoprokaryotes, phycoerythrin-rich picocyanobacteria (PE-rich) dominated in spring and summer while phycocyanin-rich picocyanobacteria (PC-rich) dominated during autumn. PE-rich and PC-rich abundances peaked during summer (1.1 × 105 and 2.0 × 105 cells mL–1) while PPE reached highest abundances in spring (1.1 × 105 cells mL–1). PPE was the main contributor to the total phytoplankton biomass (up to 73%). To assess nutrient limitation, bioassays with combinations of nitrogen (NO3 or NH4) and phosphorus additions were performed. PE-rich and PC-rich growth was mainly limited by nitrogen, with a preference for NH4 at >15°C. The three groups had distinct seasonal dynamics and different temperature ranges: 10°C and 17–19°C for PE-rich, 13–16°C for PC-rich and 11–15°C for PPE. We conclude that picophytoplankton contribute significantly to the carbon cycle in the coastal Baltic Sea and underscore the importance of investigating populations to assess the consequences of the combination of high temperature and NH4 in a future climate. [ABSTRACT FROM AUTHOR]

Picophytoplankton in the Baltic Sea includes the simplest unicellular cyanoprokaryotes (Synechococcus/Cyanobium) and photosynthetic picoeukaryotes (PPE). Picophytoplankton are thought to be a key component of the phytoplankton community, but their seasonal dynamics and relationships with nutrients and temperature are largely unknown. We monitored pico- and larger phytoplankton at a coastal site in Kalmar Sound (K-Station) weekly during 2018. Among the cyanoprokaryotes, phycoerythrin-rich picocyanobacteria (PE-rich) dominated in spring and summer while phycocyanin-rich picocyanobacteria (PC-rich) dominated during autumn. PE-rich and PC-rich abundances peaked during summer (1.1 × 105 and 2.0 × 105 cells mL–1) while PPE reached highest abundances in spring (1.1 × 105 cells mL–1). PPE was the main contributor to the total phytoplankton biomass (up to 73%). To assess nutrient limitation, bioassays with combinations of nitrogen (NO3 or NH4) and phosphorus additions were performed. PE-rich and PC-rich growth was mainly limited by nitrogen, with a preference for NH4 at >15°C. The three groups had distinct seasonal dynamics and different temperature ranges: 10°C and 17–19°C for PE-rich, 13–16°C for PC-rich and 11–15°C for PPE. We conclude that picophytoplankton contribute significantly to the carbon cycle in the coastal Baltic Sea and underscore the importance of investigating populations to assess the consequences of the combination of high temperature and NH4 in a future climate.

Phycoerythrin (PE) and phycocyanin (PC) are florescent pigments. They have the colorant role in the industry. In this study, production of PE and PC from Porphyridium cruentum were investigated at the various conditions such as different concentrations of municipal wastewater, wavelengths and salicylic acid using Response Surface Methodology-Central Composite Design (RSM-CCD), regression analysis and rstool models. The maximum RSM predicted PE concentration was 29.5 mg/g biomass at 50 % of wastewater, 510 nm of wavelength and 10 µM of salicylic acid. On the other hand, maximum RSM predicted PC concentration was 6.9 mg/g biomass at 50% of wastewater, 680 nm and 40 µM of salicylic acid. According to the ANOVA results, the square effects of the three variables (X1, X2 and X3) were found to be significant for the phycocyanin concentration, while the wastewater and salicylic acid variables (X1 and X3) were found to be important in the Phycoerythrin concentration. In addition to this, the highest PE and PC concentrations were 27.648 and 5.7104 mg/g biomass, respectively, for 50 % of wastewater, 512.5 nm and 47.0833 µM of salicylic acid according to rstool model. In conclusion, the variables such as wastewater, wavelength and salicylic acid can be used for the highest PE and PC concentration by means of RSM-CCD and rstool models and these variables may contribute to the industrial production of the two pigments. [ABSTRACT FROM AUTHOR]

The tropical red seaweed Halymenia durvillei Bory de Saint Vincent is a potential source of the high-value pigments, r-phycoerythrin (RPE) and r-phycocyanin (RPC). The unique properties of these pigments find many applications—from food to cosmetics, pharmaceuticals, and biomedical research. This study aimed to improve the land-based culture technology of H. durvillei by determining the appropriate combination of light, water motion, and stocking density that would result in high growth performance and high RPE and RPC content. Combinations of two light levels (full light, 100% and reduced light, 67%), two water motion levels (low and moderate), and three stocking densities (50, 100, and 200 g) were studied using 64.5-L glass tanks with flow-through seawater and aeration systems at the outdoor land-based seaweed nursery in Bolinao, Pangasinan, Philippines. After 6 weeks of culture, growth performance (growth rate and productivity) of H. durvillei and phycobiliprotein content were best under a combination of full light (100%), moderate water motion, and 50-g stocking density. The generally low light regime during the experimental period, which coincided with the cold, dry season characterized by shorter days and overcast skies, may have influenced H. durvillei to maximize the use of available resources such as light and possible increased nutrient availability from water motion to promote growth and phycobiliprotein synthesis. Treatments with low stocking densities generally showed satisfactory growth and phycobiliprotein content. This study provides groundwork for future research into mechanisms by which these and other factors affect the growth and physiology of H. durvillei in culture. [ABSTRACT FROM AUTHOR]

Cyanobacteria are gram-negative photosynthetic organisms and one of the most successful groups of organisms this planet has ever seen. They include some of the first life forms to evolve on Earth. During their long evolutionary history, cyanobacteria have undergone functional modifications, and these are responsible for their versatile physiology and wide ecological tolerance. The ability of cyanobacteria to tolerate high temperature, UV radiation, desiccation, and water and salt stresses contributes to their competitive success in a wide range of environments. Various species of cyanobacteria utilize different combinations of chromophores and phycobiliproteins to optimize their light-harvesting capabilities for photosynthesis. Phycobilisomes serve as the primary light-harvesting antennae for photosystem II in cyanobacteria and red algae. The aim of this research was to investigate phycobiliproteins in isolated heterocystous cyanobacteria from paddy fields in western Mazandaran. After collecting soil samples, cyanobacteria cultures were cultivated on a typical BG110. In addition to purification process, there were repeated sub-culturing on the solidified and liquid medium before the strains were characterized morphologically. Results indicated that because of adaptation of strains under accessible light condition, different species of cyanobacteria employ a high diversity of phycobiliprotein to optimize their light harvesting process in photosynthesis. Moreover, significant differences in the amounts of phycocyanin, allophycocyanin, and phycoerythrin were observed in various species. Maximum level of these protein compounds belonged to MGCY372 (Plectonema boryanum).

C-Phycocyanin (PC) and B-phycoerythrin (PE) are light-harvesting water-soluble phycobiliproteins from microalgae that belong mainly to the cyanobaceria and rhodhophytes families. Different methods have been developed for PC and PE extraction and purification from microalgae, and offer a high potential for their use as additives in sectors such as food and cosmetics. However, the main limitations of using these dyes are the sensitivity of their environmental factors, such as light fastness, temperature, and pH. We successfully employed safe lamellar nanoclays such as montmorillonite (M) and Laponite (L) for phycobiliproteins stabilization, as we did before with other natural dyes. We obtained a wide color gamut from blues to pink by combining four different factors under synthesis conditions: three dye concentrations; two laminar nanoclay sizes; a two nanoclay surface modifiers combination with cetylpyridinium bromide (CPB) and a coupling agent (3-Aminopropyl) triethoxysilane. The experimental conditions were defined according to a multilevel factorial design of experiment (DOE) to study the factors interacting in the final hybrid pigment characteristics. In both M and L, the d001 distance (nm) increased due to PC and PE adsorption. The best conditions to increase the basal space depend on the nanoclay structure, and it is better to use the surfactant for M, and silane modification for L. In addition, optical and thermal PE and PC properties significantly improved. We show the optimal synthesis conditions to increase PC and PE adsorption using the high dye concentration, with surfactant and silane depending on the nanoclay. The hybrid pigments from these phycobiliproteins offer the opportunity to perform several industrial applications, including in polymer additives, cosmetics, and packaging.

Phycobiliproteins, light-harvesting pigments found in cyanobacteria, red algae and cryptomonad are gaining importance in food, nutraceutical and pharmaceutical industries. Thus, a sustainable source of phycobiliproteins production is an essential consideration in meeting the increasing demand for these natural pigments. The present work aimed to compare the concentration and purity of phycobiliproteins from marine (Geitlerinema sp. and Synechococcus sp.) and freshwater (Oscillatoria sp. and Spirulina sp.) cyanobacteria, when subjected to different drying methods viz. sun-drying, oven-drying and freeze-drying. Results showed that the three different drying methods influenced the concentration and purity of phycobiliproteins from the different cyanobacteria. Under oven drying condition, phycocyanin concentration (mg.mL-1) was significantly higher (P < 0.05) in marine Geitlerinema sp. followed by Oscillatoria sp. Synechococcus sp. and Spirulina sp., respectively, compared to sun-drying and freezedrying methods. Phycoerythrin and allophycocyanin concentrations were also significantly higher (P < 0.05) in marine periphytic Geitlerinema sp. when compared to other cyanobacteria subjected to oven drying. In addition, results from oven-dried marine periphytic Geitlerinema sp. showed that total phycobiliproteins production and purity ratio of phycocyanin were significantly higher (P < 0.05) in comparison to sun-drying or freeze-drying Spirulina sp., Synechococcus sp. and freshwater Oscillatoria sp. [ABSTRACT FROM AUTHOR]

During the production of desalination plants, concentrated seawater is expelled to local marine environments, which may lead to hypersaline pollution. This study aimed to investigate the effects of concentrated seawater on Porphyra haitanensis by exposing P. haitanensis to salinity of 40‰, 36‰ and 30‰ (considered as the control) for 2 h, 6 h, 12 h, 24 h, 48 h and 96 h. The results showed that chlorophyll a content showed no significant change among different salinities, while carotenoid, phycocyanin and phycoerythrin contents increased in treatment with 40‰ compared with the control. Carotenoid, phycocyanin and phycoerythrin can all function as antioxidants, suggesting that hypersaline exposure might trigger oxidative stress in P. haitanensis. Overall, the present study suggests that hypersaline pollution caused by desalination plants raises significant ecological risks for local marine environments. [ABSTRACT FROM AUTHOR]

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Bone regeneration is a multistep and regular physiological process that occurs normally in fracture repair and bone defects. However, some factors such as aging, particular diseases and some drugs prevent or slowdown bone natural healing. Cell therapy using stem cells and differentiation activating factors is an effective treatment method for bone regeneration triggering in unusual conditions. Therefore, in the present study the effect of phycocyanin and phycoerythrin pigments which isolated from Spirulina platensis and Gracilaria gracilis algae was investigate on osteogenic differentiation potency of human Amniotic Mesenchymal Stem Cells (hAMSCs). For this purpose, hAMSCs were exposed to 300, 500, and 700 µg/ml concentrations of phycocyanin and phycoerythrin pigments and then the cells viability was measured with MTT assay in 48 and 72 h after treatment. The osteo-differentiation level of cells was studied by measuring ALP activity using calorimetric method and Alizarin red staining for calcium deposition in 7 and 21 days after treatment. Also, total RNA of cells was extracted in different time periods and then cDNA synthesized with specific primers, and relative expression of Runx2 , β-catenin and Osteocalcin genes were investigated using SYBR Green RT-qPCR technique. Osteogenic differentiation of hAMSCs that treated with pigments was confirmed by mineral deposits staining and increased level of ALP activity. Furthermore, these pigments elevated significantly the expression of osteogenic marker genes compared to control samples and caused hAMSCs to differentiate into osteoblast cells. According to these results, phycocyanin and phycoerythrin may suggest as suitable osteogenic supplements with low toxicity, low cost and high efficiency, although the molecular mechanism of its efficacy is not available yet. [Display omitted] • Algal phycobiliproteins increased significantly osteogenic marker genes in hAMSCs. • Phycobiliprotein supplemented culture media induced calcium deposition on 21 days. • Phycoerythrin was more effective for osteoinduction compared with phycocyanin. [ABSTRACT FROM AUTHOR]

The interest of the food industry in replacing artificial dyes with natural pigments has grown recently. Cyanobacterial phycobiliproteins (PBPs), phycoerythrin (PE) and phycocyanin (PC), are colored water-soluble proteins that are used as natural pigments. Additionally, red PE and blue PC have antioxidant capabilities. We have formulated a new food prototype based on PBP-fortified skim milk. PBPs from Andean cyanobacteria were purified by ammonium sulfate precipitation, ion-exchange chromatography, and freeze-drying. The stability of PE and PC was evaluated by changes in their absorption spectra at various pH (1−14) and temperature (0−80 °C) values. Purified PBPs showed chemical stability under pH values of 5 to 8 and at temperatures between 0 and 50 °C. The antioxidant property of PBP was confirmed by ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical ion scavenging, and FRAP (Ferric Antioxidant Power) assays. The absence of PBP toxicity against Caenorhabditis elegans was confirmed up to 1 mg PBP/mL. Skim milk fortified with PE obtained a higher score after sensory tests. Thus, a functional food based on skim milk-containing cyanobacterial PBPs can be considered an innovative beverage for the food industry. PBPs were stable at an ultra-high temperature (138 °C and 4 s). PBP stability improvements by changes at its primary structure and the incorporation of freeze-dried PBPs into sachets should be considered as alternatives for their future commercialization.

Five red algae, Agardhiella subulata, Gracilariopsis longissima, Gracilaria vermiculophylla, Polysiphonia morrowii, and Pyropia elongata were sampled in winter for the extraction of phycoerythrin. The extracted phycoerythrin molecules were compared with the commercial phycoerythrin to determine the intrinsic fluorescence characteristics of the single pigments. An improved selective method for phycoerythrin extraction was set up for routinary investigation. The use of the mortar and pestle method for tissue homogenization with a freeze-thawing cycle allowed a simple and complete homogenization of the red algae. The extraction of phycoerythrin with diluted EDTA solutions (1 mM) at pH 9 enabled a selective and easy extraction of the pigment with 95-98% extraction efficiency. The way pH affected the phycoerythrin, phycocyanin, and allophycocyanin selective extraction was also evaluated. The 3D fingerprint of each pigment was recorded, and a comparison of different phycoerythrin spectra was performed by fluorescence spectroscopy highlighting differences in A. subulata and P. morrowii phycoerythrins in comparison with commercial standards purified from Pyropia. The productivity and the advantages of phycoerythrin that was extracted from unattached red algal species are discussed. [ABSTRACT FROM AUTHOR]

Phycobilisomes are light-harvesting protein complexes and are widely distributed in red algae and cyanobacteria. Each phycobilisome contains highly fluorescent protein components called phycobiliproteins. Based upon the distinct physiochemical properties, phycobiliproteins are classified as allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin. In the present study, we describe purification and structural characterization of a novel phycocyanin and phycoerythrin isolated from a marine red macroalga, Centroceras clavulatum . The absorbance and fluorescence studies indicated that the purified proteins belong to R-Phycocyanin (R-PC) and R-Phycoerythrin (R-PE). The single bands under native-polyacrylamide gel electrophoresis revealed the intact molecular weights of R-PC and R-PE as 110 kDa and 250 kDa. The polypeptide compositions of the two proteins were demonstrated by SDS-PAGE. The result showed that R-PC contains two bands at 17 and 21 kDa and were identified as α and β subunits through mass spectrometry based proteomics experiments. SDS-PAGE of R-PE showed three distinct bands at 18, 19 and 35 kDa and was subsequently identified as α, β and γ subunits. The near-complete amino acid sequences of α and β subunits of R-PC and R-PE were derived from mass spectrometric data combined with Mascot software and multiple de novo sequencing tools followed by homology search and manual validation. [ABSTRACT FROM AUTHOR]