Recent advances in production, purification and applications of phycobiliproteins.

An obligatory sunlight requirement for photosynthesis has exposed cyanobacteria to different quantity and quality of light. Cyanobacteria can exhibit efficient photosynthesis over broad region (450 to 650 nm) of solar spectrum with the help of brilliantly coloured pigment proteins called phycobiliproteins (PBPs). Besides light-harvesting, PBPs are found to involve in several life sustaining phenomena including photoprotection in cyanobacteria. The unique spectral features (like strong absorbance and fluorescence), proteineous nature and, some imperative properties like hepato-protective, anti-oxidants, anti-inflammatory and anti-aging activity of PBPs enable their use in food, cosmetics, pharmaceutical and biomedical industries. PBPs have been also noted to show beneficial effect in therapeutics of some disease like Alzheimer and cancer. Such large range of applications increases the demand of PBPs in commodity market. Therefore, the large-scale and coast effective production of PBPs is the real need of time. To fulfil this need, many researchers have been working to find the potential producer of PBPs for the production and purification of PBPs. Results of these efforts have caused the inventions of some novel techniques like mixotrophic and heterotrophic strategies for production and aqueous two phase separation for purification purpose. Overall, the present review summarises the recent findings and identifies gaps in the field of production, purification and applications of this biological and economically important proteins.

Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin, Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM.

An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with α- and ß-subunit of PE and APC occurred after 4 h of UV-B exposure. After 5 h of UV-B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV-A radiation, a severe decrease in fluorescence of all PBPs was observed under UV-B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15-30 min of UV-B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV-B radiation. Moreover, the present study indicates that UV-B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.

Physico-chemical factors affecting the in vitro stability of phycobiliproteins from Phormidium rubidum A09DM.

The functionality and stability of phycobiliproteins (PBPs) phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC) were investigated under various temperatures, pHs and oxidative stressors. All PBPs were thermostable up to 4-40°C; however, their concentration decreased rapidly at 60-80°C. The maximum stability of all PBPs was in the pH range 6.0-7.0. Decrease in PBPs content was found under high acidic (pH 2-4) and alkaline conditions (pH 8-12). The oxidizing agent (0.1-0.6%) showed the least effect on the stability of PBPs; however, 0.8-1.0% H2O2 caused significant loss of PBPs. Contrary to PE, PC and APC was more susceptible to an oxidizing agent. The chromophore associated with α- and ß-subunit of PBPs and thus, their functionality (fluorescence) was severely affected under high temperature (60-80°C), and oxidizing agent, as well as low (2-4) and high (8-12) pH. Contrary to PC and APC, functionality of PE was surprisingly maintained even at pHs 6-12 and under oxidative stress.

Cyanobacterial Sunscreen Scytonemin: Role in Photoprotection and Biomedical Research.

Cyanobacteria are the most promising group of photosynthetic microorganisms capable of producing an array of natural products of industrial importance. Scytonemin is a small hydrophobic alkaloid pigment molecules present in the extracellular sheath of several cyanobacteria as a protective mechanism against short wavelength solar ultraviolet (UV) radiation. It has great efficacy to minimize the production of reactive oxygen species and formation of DNA lesions. The biosynthesis of scytonemin is regulated by different physico-chemical stressors. Scytonemin display multiple roles, functioning as a potent UV sunscreen and antioxidant molecules, and can be exploited in cosmetic and other industries for the development of new cosmeceuticals. Herein, we review the occurrence, biosynthesis, and potential application of scytonemin in photoprotection, pharmaceuticals, and biomedical research.

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM.

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and ß- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and ß- subunits (known as αß monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αß monomers in solution and in crystal lattice. The overall tertiary structures of α- and ß- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein.

A stable and functional single peptide phycoerythrin (15.45 kDa) from Lyngbya sp. A09DM.

A functional and stable truncated-phycoerythrin (T-PE) was found as a result of spontaneous in vitro truncation. Truncation was noticed to occur during storage of purified native-phycoerythrin (N-PE) isolated from Lyngbya sp. A09DM. SDS and native-PAGE analysis revealed the truncation of N-PE, containing α (19.0 kDa)--and ß (21.5 kDa)--subunits to the only single peptide of ∼15.45 kDa (T-PE). The peptide mass fingerprinting (PMF) and MS/MS analysis indicated that T-PE is the part of α-subunit of N-PE. UV-visible absorption peak of N-PE was found to split into two peaks (540 and 565 nm) after truncation, suggesting the alterations in its folded state. The emission spectra of both N-PE and T-PE show the emission band centered at 581 nm (upon excitation at 559 nm) suggested the maintenance of fluorescence even after significant truncation. Urea-induced denaturation and Gibbs-free energy (ΔGD°) calculations suggested that the folding and structural stability of T-PE was almost similar to that of N-PE. Presented bunch of evidences revealed the truncation in N-PE without perturbing its folding, structural stability and functionality (fluorescence), and thereby suggested its applicability in fluorescence based biomedical techniques where smaller fluorescence molecules are more preferable.

The phycobilisomes: an early requisite for efficient photosynthesis in cyanobacteria.

Cyanobacteria trap light energy by arrays of pigment molecules termed "phycobilisomes (PBSs)", organized proximal to "reaction centers" at which chlorophyll perform the energy transduction steps with highest quantum efficiency. PBSs, composed of sequential assembly of various chromophorylated phycobiliproteins (PBPs), as well as nonchromophoric, basic and hydrophobic polypeptides called linkers. Atomic resolution structure of PBP is a heterodimer of two structurally related polypeptides but distinct specialised polypeptides- a and ß, made up of seven alpha-helices each which played a crucial step in evolution of PBPs. PBPs carry out various light dependent responses such as complementary chromatic adaptation. The aim of this review is to summarize and discuss the recent progress in this field and to highlight the new and the questions that remain unresolved.

Phormidium phycoerythrin forms hexamers in crystals: a crystallographic study.

The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Šresolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, ß = 82.50, γ = 60.34°. The molecular-replacement solution confirmed the presence of 12 αß monomers in the P1 cell. The Phormidium PE elutes as an (αß)3 trimer of αß monomers from a molecular-sieve column and exists as [(αß)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher-order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.

Phycoerythrin extends life span and health span of Caenorhabditis elegans.

In the present study, we tested the antioxidant activity of phycoerythrin (PE, an oligomeric light harvesting protein isolated from Lyngbya sp. A09DM) to curtail aging effects in Caenorhabditis elegans. Purified PE (100 µg/ml) dietary supplement was given to C. elegans and investigated for its anti-aging potential. PE treatment improved the mean life span of wild type (N2)-animals from 15 ± 0.1 to 19.9 ± 0.3 days. PE treatment also moderated the decline in aging-associated physiological functions like pharyngeal pumping and locomotion with increasing age of N2 worms. Moreover, PE treatment also enhanced the stress tolerance in 5-day-aged adults with increase in mean survival rate from 22.2 ± 2.5 to 41.6 ± 2.5% under thermo stress and from 30.1 ± 3.2 to 63.1 ± 6.4% under oxidative (hydrogen peroxide)-stress. PE treatment was also noted to moderate the heat-induced expression of human amyloid-beta(Aß1-42) peptide and associated paralysis in the muscle tissues of transgenic C. elegans CL4176 (Alzheimer's disease model). Effectiveness of PE in expanding the life span of mutant C. elegans, knockout for some up (daf-2 and age-1)- and down (daf-16)-stream regulators of insulin/IGF-1 signaling (IIS), shows the independency of PE effect from DAF-2-AGE-1-DAF-16 signaling pathway. Moreover, the inability of PE in expanding the life span of hsf-1 knockout C. elegans(sy441) suggests the dependency of PE effect on heat shock transcription factor (HSF-1) controlling stress-induced gene expression. In conclusion, our results demonstrated a novel anti-aging activity of PE which conferred increased resistance to cellular stress resulting in improved life span and health span of C. elegans.