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, antioxidants, 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.

EXCITATION ENERGY TRANSFER IN VITRO BETWEEN PHYCOBILIPROTEINS AND THYLAKOID PHOTOSYSTEM Ⅱ OF HIGHER PLANTS

The excitation energy transfer from phycobiliproteins to thylakoid PSII of higher plants was investigated. When incubated with spinach thylakoids, phycobiliproteins isolated from red and blue- green algae transferred light energy absorbed to spinach PSII. The efficiency of energy transfer was dependent on the kind of phycobiliproteins used. If spinach thylakoids were replaced by the thylakoids of Brassica chinensis, R phycoerythin or C- phycocyanin did not transfer their excitation energy to PSII of Brassica chinensis unless allophycocyanin was present.

Computer Simulation on Kinetics of Primary Processes in Photosynthesis Ⅰ——Mechanism of Exciton Annihilation of Excited State Decay in Phycobiliproteins

A discrete model was used for studying the effects of exciton annihilation on the decay behavior of the excited state in phycobiliprotein super-molecules. The simulation shows: The more the chromophores are and the higher the excitation beam intensity is, the more susceptible to annihilation is a system; annihilation starts to occur at 10^(13) photons/cm^2 and becomes saturated at about 10^(15)—10^(16) photons/cm^2 with the parameters used in the current work. Whether the exciton annihilation occurs or not should not be simply judged from whether the decaying data could be fitted with a mono-exponential function. The quantum yield of fluorescence decreases synchronously with the increase of exciton annihilation extent. The so called 'safety intensity' has significance only in special cases. In fact, annihilation extent is dependent not only on the pulse intensity but also on the width of the pulse, the optical cross section of a chromophore as well as the light absorption coefficient of a system.

Studies on kinetics of reactions between phycobiliproteins and hydroxyl radicals by a pulse radiolytic technique

Scavenging of hydroxyl radical (·OH) by phycobiliproteins (C-phycocyanin, Allophyco- cyanin and R-phycoerythrin) was studied by competitive kinetics methods. Hydroxyl radicals were generated from pulse radiolysis of aqueous systems saturated with nitrous oxide (N2O). The experimental results indicated that the three types of phycobiliproteins are all strong scavengers of· OH, the rate constants are around (2.8-5.6)×109 L·2 mol1 · s 1.

Bioinformatics Analysis on α Subunit Gene of Phycobiliprotein from Spirulina maxima

[Objective]The α subunit gene of phycobiliprotein from Spirulina maxima was studied in order to provide a basis for the subsequent study of phycobiliprotein.[Method] Amino acids composition,signal peptides,hydrophobicity/hydrophilicity and trails-membrane topological structure of α subunit gene of phycobiliprotein from Spirulina maxima which registered in GenBank（GenBank AF441177） were analyzed and predicted by the tools of bioinformatic analysis.Meanwhile,phylogenetic tree was constructed based on α subunit gene of phycobiliprotein from Spirulina maxima,and its molecular evolution was also analyzed.[Result]The phycobiliprotein was rich in amino acids,which not only contained 18 kinds of essential amino acids,but also contained some non-essential amino acids like glycine,aspartic acid,etc.;Analysis on signal peptides and trails-membrane topological structure showed that the phycobiliprotein belonged to intracellular protein;Analysis on hydrophobicity/hydrophilicity showed that the phycobiliprotein belonged to hydrophilic protein;Phylogenetic analysis showed that the phycobiliprotein had a high homology with Arthrospira,which reached 99%-100%.[Conclusion]The study provided a certain reference for studying the relationship and interaction between α subunit and β subunit.

Comparative analysis of four essential Gracilariaceae species in China based on whole transcriptomic sequencing

Three Gracilaria species, G. chouae, G. blodgettii, G. vermiculophylla and a close relative species, Gracilari-opsis lemaneiformis which is now nominated as Gracilaria lemaneiformis, are the typically indigenous spe-cies which are important resources for the production of special proteins, phycobilisomes, special carbo-hydrates, and agar in China. In this study, de novo transcriptome sequencing on these four species using the next generation sequencing technology was performed for the first time. Functional annotations on assembled sequencing reads showed that the transcriptomic profiles were quite different between G. lema-neiformis and other three Gracilaria species. Comparative analysis of differential gene expression related to carbohydrate and phycobiliprotein metabolisms also showed that the expression profiles of these essential genes were different in four species. The genes encoding allophycocyanin, phycocyanin and phycoerythrin were further examined in four species and their deduced amino acid sequences were used for phylogenetic analysis to confirm that G. lemaneiformis had close relationship to genus Gracilaria, as well as that within genus Gracilaria, G. chouae had closer relationship to G. vermiculophylla rather than to G. blodgettii. The de novo transcriptome study on four species provided a valuable genomic resource for further understanding and analysis on biological and evolutionary study among marine algae.

Long term diazotrophic cultivation induces phycobiliprotein production in Anabaena variabilis IMU8

Cyanobacteria are considered as a sustainable feedstock for the production of biochemically active compounds such as phycobiliproteins(PBPs).In this study,the impact of nitrogen(N)and phosphorus(P)availability on PBP production of“N-free acclimated”Anabaena variabilis IMU8 was analyzed.Upon isolation and identification,the cyanobacterium has been maintained in N-free BG-11 medium for more than 20 months.For experimentation,the strain was incubated in N-replete,N-depleted,N-P-depleted BG-11 medium.Long-term diazotrophic cultivation of A.variabilis IMU8 resulted in elevated PBP productivity with a limited impact on growth.When compared to N-depleted ones,N supply stimulated a slight induction of growth and total saccharide production,but total protein content did not change while PBP production decreased.On the other hand,N-P-depletion resulted in decreased growth rate along with reduced total protein and PBP production while rapid induction of total saccharide production was recorded.Fourier transform infrared spectroscopy results refer that membrane-bound oligosaccharides may have regulatory roles for PBP production in A.variabilis IMU8 during long term diazotrophic cultivation.

Heterogenous Expression of Synechocystis sp. PCC 6803 Deg Proteases and Their Possible Roles on Phycobiliprotein Degradation in vitro

The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA （degP, slr1204）, hhoA （degQ, sll1679） and hhoB （degS, sll1427）. This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and phycobiliproteins in vitro, which implicated that they were proteases possibly involved in phycobiliprotein degradation in vivo.

Effect of Iron on Growth, Pigmentation and Antioxidative Activity of Bloom Forming Cyanobacteria

Cyanobacterial blooms are ubiquitous in fresh and brackish eutrophic waters in India. The cyanotoxins produced by many bloom forming cyanobacteria severely affect the health of animals, fishes, birds and human beings. Different physical and chemical factors contribute towards bloom formation. Ten bloom forming cyanobacteria were isolated from natural blooms of northern India. The strains were purified and enriched in the laboratory. The aim of this study was to understand the influence of iron on growth, pigmentation and antioxidative activity of enzymes-catalase and ascorbate peroxidase of bloom forming cyanobacteria. Results show that different strains of bloom forming cyanobacteria attain optimum growth at varied concentration of iron. The cyanobacterial strains like Synechocystis aquatalis, Merismopedia glauca, Anabaena variabilis and Anabaena iyengarii exhibit maximum growth at low iron concentration （2 pM） while some species require higher concentration of iron for their optimum growth namely, Arthrospira platensis show optimum growth at 10uM, and Nostocpaludosum shows maximum growth at 100uM concentration of iron. It was also noticed that chlorophyll and phycobiliprotein content also varies with change in iron concentration. The activity of antioxidative enzymescatalase and ascorbate peroxidase was noticed in all ten cyanobacterial strains. In the light of the findings, it seems that Arthrospira platensis possess maximum catalase and ascorbate peroxidase activity. Increment in concentration beyond optimum value leads to deterioration in the growth, pigment content and enzymatic activity of the cyanobacterial strains. Knowledge about the factors influencing growth of bloom forming cyanobacteria will help to work out ways for eradication of hazardous cyanobacterial blooms.

Progress in the study on structure and function of the antenna of algae

IN the evolution series, algae possess a position between green plants and photosynthetic bacteria. The photosynthetic apparatus of algae and plants are very similar because both of them contain the two phótosynthetic reaction centers of PSⅡ and PSⅠ and have the same photosynthetic function. Therefore, study of the photosynthesis of algae has a common significance. On the other hand, the light-harvesting antennae of plants are chlorophyll a/b-protein complex in thylakoids while those of algae are the phycobilisomes. A phycobilisome is a large super-

RESONANCE RAMAN SCATTERING OF PHYCO BILISOMES AND PHYCO BILIPROTEINS FROM CYANOBACTERIUM Westiellopsis prolifica

Phycobilisome is a kind of supramolecular light-harvesting pigment protein complex, which can facilitate the absorption and transfer of light energy to the photosynthetic reaction centers in cyanobacteria and red algae. The phycobilisomes (hereafter called PBS)

Molecular mechanisms for interaction of glycine betaine with supra-molecular phycobiliprotein complexes

Glycine betaine(GB) is a biologically important small molecule protecting cells,proteins and enzymes in vivo and in vitro under environmental stresses.Recently,it was found that GB could also relax the structure and inactivate the function of phycobiliproteins and phycobilisome(PBS),a kind of supramolecular complexes,in cyanobacterial cells.The molecular mechanisms for the opposite phenomena are quite ambiguous.Taking PBS and a trimeric or monomeric C-phycocyanin(C-PC) as models,the molecular mechanism for the interaction of GB with supra-molecular complexes or nuclear proteins was investigated.The energetic decoupling of PBS components induced by GB suggests that the PBS core-membrane linking polypeptide was the most sensitive site while the rod-core linker was the next.Biochemistry analysis proves that PBS structure was loosened but not dissociated into the components.On the basis of the results and structure knowledge,it was proposed that GB screened the electrostatic attraction of the opposite charges on a linker and a protein leading to a much looser structure.It was observed that GB induced a spectral blue shift for trimeric C-PC but a red shift for a monomeric C-PC(a nuclear protein),which were ascribed to GB's screening of the electrostatic attraction of a linker to a protein and strengthening of the hydrophobic interaction between C-PC monomers.The trimers and monomers' forming of the same products under high concentration of GB was ascribed to a compromise of the opposite interaction forces.

Phytochemical screening of twelve species of phytoplankton isolated from Arabian Sea coast

Objective:To analyze the phytochemicals in twelve species of marine phytoplankton.Methods:Total phenolic content of methanol extract was estimated by the Folin-Ciocalteu method.Total flavonoid content of the methanol extarct was determined by aluminium chloride method.Chlorophylls,β-carotene and astaxanthin were estimated by acetone extraction method.Vitamin C was determined by dinitrophenyl-hydrazine method.Phycobiliproteins such as allophycocyanin,phycocyanin and phycoerythrin in the aqueous extracts were determined.Results:Total phenolics varied from 5.41 mg gallic acid equivalents/g dry weight(DW)in Phormidium corium(P.corium)to 17.37 mg gallic acid equivalents/g DW in Oscillatoria fremyii(O.fremyii).Total flavonoids ranged between 0.74 mg quercetin equivalent/g DW in P.corium and 9.87 mg quercetin equivalent/g DW in Nannochloropsis oceanica.Chlorophyll-a pigment was high in Chaetoceros calcitrans(C.calcitrans)(15.51 mg/g DW)and low in P.corium(1.08 mg/g DW).Chlorophyll-c ranged between 0.07 mg/g DW in Nannochloropsis oceanica and 4.62 mg/g DW in C.calcitrans.High contents ofβ-carotene and astaxanthin were found in C.calcitrans and low in P.corium which ranged from 0.33 to 10.03 mg/g DW and 0.18 to 3.85 mg/g DW,respectively.Vitamin C content varied from 0.50 mg/g DW in C.calcitrans to 1.51 mg/g DW in Phormidium tenue.O.fremyii showed highest total phycobiliproteins of 317.05 mg/g DW.High contents of allophycocyanin and phycocyanin were found in O.fremyii,whereas high contents of phycoerythrin were found in Oscillatoria sancta.All the three phycobiliproteins were low in Chroococcus turgidus.Conclusions:Marine phytoplankton are one of the natural sources providing novel biologically active compounds with potential for pharmaceutical applications.

Preliminary crystallographic investigation of allophycocyanin from Porphyra yezoensis in cubic crystal form

THE phycobilisomes in blue-green algae and red algae are the functional units which absorb and transfer light energy. These supramolecular aggregates are attached on the outer surface of the thylakoid or photosynthetic membrane. Phycobilisomes absorb light in the range of 450—650 nm with an overall quantum efficiency of almost 100%, Electron microscopic studies showed that phycobilisomes consist of two distinct structure parts: the core and the rods.