Phycocyanin Subunits Research Articles

Studying the effect of magnetic fields on biological activity of phycocyanin extracted from Desmonostoc alborzicum

Multiple studies have provided evidence of the positive effects of treatment with magnetic fields on the efficiency of producing secondary compounds, development, and the overall condition of cyanobacteria cultures. Therefore, the main objective of this study is to evaluate the impact of different magnetic fields (30 and 60 mT for 21 days) on the biological activity of phycocyanin (PC) extracted from Desmonostoc alborzicum. Results showed that a 30 mT magnetic field had increased the concentration and purity of PC. However, the highest antioxidant and antibacterial and antifungal activity was found in the PC exposed to 60 mT magnetic field (p<0.05). However the results of SDS-PAGE gel showed that the amounts of alpha and beta subunits of PC treated with magnetic fields of 30 mT rose by 1.4 and 1.6 times, respectively, compared to the control. In conclusion, applying a 60 mT magnetic field could have the highest biological activity for PC in comparison to 30 mT.

Interactome Analysis of ClpX Reveals Its Regulatory Role in Metabolism and Photosynthesis in Cyanobacteria.

Protein homeostasis is essential for cyanobacteria to maintain proper cellular function under adverse and fluctuating conditions. The AAA+ superfamily of proteolytic complexes in cyanobacteria plays a critical role in this process, including ClpXP, which comprises a hexameric ATPase ClpX and a tetradecameric peptidase ClpP. Despite the physiological effects of ClpX on growth and photosynthesis, its potential substrates and underlying mechanisms in cyanobacteria remain unknown. In this study, we employed a streptavidin-biotin affinity pull-down assay coupled with label-free proteome quantitation to analyze the interactome of ClpX in the model cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We identified 503 proteins as potential ClpX-binding targets, many of which had novel interactions. These ClpX-binding targets were found to be involved in various biological processes, with particular enrichment in metabolic processes and photosynthesis. Using protein-protein docking, GST pull-down, and biolayer interferometry assays, we confirmed the direct association of ClpX with the photosynthetic proteins, ferredoxin-NADP+ oxidoreductase (FNR) and phycocyanin subunit (CpcA). Subsequent functional investigations revealed that ClpX participates in the maintenance of FNR homeostasis and functionality in Synechocystis grown under different light conditions. Overall, our study provides a comprehensive understanding of the extensive functions regulated by ClpX in cyanobacteria to maintain protein homeostasis and adapt to environmental challenges.

Combination of Mechanical/Physical Pretreatments with Trypsin or Pancreatin on Arthrospira platensis Protein Degradation

Arthrospira platensis contains high protein content and quality, which makes it a promising feed source for livestock animals. However, this microalga presents a recalcitrant peptidoglycan cell wall, and its main proteins form protein-pigment complexes attached to the algal thylakoid membrane. The purpose of the present study was to evaluate the effect of mechanical/physical pretreatments (bead milling, extrusion, freeze-drying, heating, microwave and sonication) combined with commercial enzymes (trypsin or pancreatin) on the degradation of A. platensis proteins. Protein degradation was assessed through the quantification of total protein and protein fractions (18–26 kDa, 40–48 kDa and others) on SDS-PAGE gels and the evaluation of the coefficient of protein degradation (CPD). The results showed that no significant differences were found among treatments for CPD values, except for an increase related to 18–26 kDa protein (phycocyanin subunits) with extrusion combined with pancreatin. In addition, extrusion and microwave caused a decrease of total protein in algal supernatant probably due to a denaturation/aggregation and reduction of solubility. Overall, extrusion is a promising pretreatment for A. platensis cell wall disruption and protein bioaccessibility. Further studies could elucidate how the effect of extrusion on protein solubility affects the activity of peptidases on protein degradation.

A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes.

Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring‐shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light‐harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.

Phycocyanin fluorescent probe from Arthrospira platensis: preparation and application in LED-CCD fluorescence density strip qualitative detection system

Phycocyanin (PC) is a natural fluorescent protein which is widely used in medical diagnosis, biology, biotechnology, foods, and medicine. However, the disadvantages in traditional purification methods, such as complicated preparation process, low yield, inadequate quantity, and high price, limit its range of application. In this study, we established a rapid purification method suitable for large-scale production to prepare PC from Arthrospira platensis. The membrane separation and two-stage ammonium sulfate precipitation yielded PC with a purity ratio (A620nm/A280nm) of 3.65. This purification method of PC proved to be efficient and cost-effective compared with previously reported methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the molecular weight of α and β subunits of PC was 16.6 and 21.2 kDa. The main molecular weight of intact PC determined through high-performance size exclusion chromatography (HPSEC) was around 91 kDa, indicating that the PC from A. platensis existed mainly in the form of (αβ)3 at pH 7.0. The purified PC fluorescent probe was compatible for light-emitting diode (LED)—charge-coupled device (CCD) fluorescence density strip qualitative detection system. A relatively sensitive novel fluorescent test method was developed for the quantitative detection of PC showing a linear response to PC within the range of 0.07–3.64 μM, and with a limit of detection of 0.034 μM. This method could provide expedient quantitative information and showed great potential for rapid detection in environmental and food safety research.

Complementary chromatic and far-red photoacclimations in Synechococcus ATCC 29403 (PCC 7335). I: The phycobilisomes, a proteomic approach.

Synechococcus ATCC 29403 (PCC 7335) is a unicellular cyanobacterium isolated from Puerto Peñasco, Sonora Mexico. This cyanobacterium performs complementary chromatic acclimation (CCA), far-red light photoacclimation (FaRLiP), and nitrogen fixation. The Synechococcus PCC 7335 genome contains at least 31 genes for proteins of the phycobilisome (PBS). Nine constitutive genes were expressed when cells were grown under white or red lights and the resulting proteins were identified by mass spectrometry in isolated PBS. Five inducible genes were expressed under white light, and phycoerythrin subunits and associated linker proteins were detected. The proteins of five inducible genes expressed under red light were identified, the induced phycocyanin subunits, two rod linkers and the rod-capping linker. The five genes for FaRLiP phycobilisomes were expressed under far-red light together with the apcF gene, and the proteins were identified by mass spectrometry after isoelectric focusing and SDS-PAGE. Based on in silico analysis, Phylogenetic trees, and the observation of a highly conserved amino acid sequence in far-red light absorbing alpha allophycoproteins encoded by FaRLiP gene cluster, we propose a new nomenclature for the genes. Based on a ratio of ApcG2/ApcG3 of six, a model with the arrangement of the allophycocyanin trimers of the core is proposed.

Structural characterization and antioxidant potential of phycocyanin from the cyanobacterium Geitlerinema sp. H8DM

We are reporting characterization of phycocyanin (PC), a prime light harvesting pigment, composed of nearly the same molecular weight of alpha- (17.5 kDa) and beta- (18.1 kDa) subunits in Geitlerinema sp. H8DM. Phycocyanin was purified using ammonium sulfate and anionic exchange chromatography. The fluorescence emission spectrum of PC was 637 nm when excited over 589 nm, which denoted integrity and functionality of protein structure. PC was depicted as a single subunit of ~18 kDa based on SDS-PAGE. However, similarity between both subunits was proven by two spots on gel using two dimensional gel electrophoresis. MALDI ToF/ToF showed structure similarities with alpha and beta subunits of PC. Urea induced Gibbs free energy denoted that folding and structural stability of this pigment is similar to phycocyanin of other species. Biophysical properties of peptide sequence were analyzed for several parameters and 3D model for its correlation as antioxidant. Furthermore, verification of in-silico data of PC was done by in vitro anti-oxidant assays. Hence, due to high availability, stability and antioxidant properties it can be used for auxiliary applications as colorant and therapeutic agent against oxidative stress.

Solution structure and excitation energy transfer in phycobiliproteins of Acaryochloris marina investigated by small angle scattering

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

Excitation energy transfer and electron-vibrational coupling in phycobiliproteins of the cyanobacterium Acaryochloris marina investigated by site-selective spectroscopy

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) — containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635nm, 645nm, 654nm, 659nm and a terminal emitter at about 673nm. The electronic states at ~635 and 645nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37cm−1, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634cm−1 appear to reflect CNH+ and CC stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BackgroundThe production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.ResultsThe transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.ConclusionsChanges in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.

Isolation and characterization of thermostable phycocyanin from Galdieria sulphuraria

Phycocyanin is a highly valuable pigmented protein synthesized by several species of cyanobacteria and red alga. In this study we demonstrate the production of thermostable phycocyanin from the unicellular red alga Galdieria sulphuraria. Phycocyanin was extracted by repeated freeze-thaw cycles and purified in a two-step process using ammonium sulfate fractionation, at 25% and 50% concentrations. Purified phycocyanin exhibited maximum absorbance at 620 nm, and the purity ratio (A620/A280) was found to be greater than 4. The recovery efficiency of phycocyanin from the crude extract was above 80%. In total, approximately 19 milligram pure phycocyanin was obtained from 3 g of wet cell mass of Galdieria sp. Subunits α and β of the protein were separated by SDS-PAGE and analyzed by MALDITOF mass spectrometry for identification, which confirmed that the isolated protein is phycocyanin. The molecular weight of α and β subunits of phycocyanin was found to be 17.6 and 18.4 kDa, respectively.

Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421

Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

Reduced light-harvesting antenna: Consequences on cyanobacterial metabolism and photosynthetic productivity

Cyanobacteria are potential candidates for future photobiological hydrogen production. For this purpose, optimization of cyanobacterial metabolism and up-regulation of the linear electron flow are mandatory. One strategy to achieve this goal is the reduction of the photosynthetic antenna size. Here, we characterize the photosynthetic performance of two Synechocystis PCC 6803 antenna mutants in respect to culture density and light intensity under well-defined − continuous − cultivation conditions. The metabolic state of the mutants is defined by spectroscopic investigations and an in-depth proteomic analysis.Our results show that both biotic (i.e. balanced photosystem 2 to photosystem 1 ratios, light tolerance) and abiotic parameters (i.e. light intensity, cell density) are important for the optimization of photosynthetic efficiency which, in turn, is a prerequisite for high-yield photobiological hydrogen production. While a complete loss of light-harvesting antenna – as observed in the PAL mutant – has a significant negative impact on robustness and fitness, the Olive mutant lacking only the phycocyanin subunits reaches higher cell densities in our photobioreactor setup. This results in higher time-space-yields. For this reason the Olive mutant is a promising candidate for the design of future hydrogen production.

Stable isolation of phycocyanin from Spirulina platensis associated with high-pressure extraction process.

A method for stably purifying a functional dye, phycocyanin from Spirulina platensis was developed by a hexane extraction process combined with high pressure. This was necessary because this dye is known to be very unstable during normal extraction processes. The purification yield of this method was estimated as 10.2%, whose value is 3%–5% higher than is the case from another conventional separation method using phosphate buffer. The isolated phycocyanin from this process also showed the highest purity of 0.909 based on absorbance of 2.104 at 280 nm and 1.912 at 620 nm. Two subunits of phycocyanin namely α-phycocyanin (18.4 kDa) and β-phycocyanin (21.3 kDa) were found to remain from the original mixtures after being extracted, based on SDS-PAGE analysis, clearly demonstrating that this process can stably extract phycocyanin and is not affected by extraction solvent, temperature, etc. The stability of the extracted phycocyanin was also confirmed by comparing its DPPH (α,α-diphenyl-β-picrylhydrazyl) scavenging activity, showing 83% removal of oxygen free radicals. This activity was about 15% higher than that of commercially available standard phycocyanin, which implies that the combined extraction method can yield relatively intact chromoprotein through absence of degradation. The results were achieved because the low temperature and high pressure extraction effectively disrupted the cell membrane of Spirulina platensis and degraded less the polypeptide subunits of phycocyanin (which is a temperature/pH-sensitive chromoprotein) as well as increasing the extraction yield.

Epizoic cyanobacteria associated with a Neotropical harvestman (Opiliones: Sclerosomatidae) from Costa Rica

Abstract We describe multiple observations of epizoic cyanobacteria occurring on external surfaces of a species of sclerosomatid harvestman (Prionostemma sp.) in Costa Rica. In the field we collected four adults (three males, one female) that had green films growing upon the dorsal surfaces of the carapace and abdominal scutum. Examination by SEM revealed dense clusters of what appeared to be small prokaryotic cells (1–5 µm in diameter) covering the external surfaces of the carapace, abdominal scutum and coxae. We extracted DNA from the films of two specimens. The DNA was used as a template to amplify the intergenic spacer region (IGS) between the beta and alpha phycocyanin subunits (a signature DNA sequence, unique to cyanobacteria) by PCR. We successfully amplified an approximately 700 base pair product using DNA extracted from the film and did not obtain any product from the harvestman lacking the film. Our observation represents the second confirmed occurrence of epizoic cyanobacteria on Neotropical h...

High-Resolution Crystal Structures of Trimeric and Rod Phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (LR8.7), the rod linker (LR), and only one of three rod–core linkers (LRC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the LR protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both alpha- and beta-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species.

A novel method of single step hydrophobic interaction chromatography for the purification of phycocyanin from Phormidium fragile and its characterization for antioxidant property

Phycocyanin – a major phycobiliprotein constitutively produced by many cyanobacteria hold several promising applications in diagnostics, biomedical research, and therapeutics. This paper discusses a novel rapid method for the purification of cyanobacterial phycocyanin (C-PC) from Phormidium fragile using hydrophobic interaction chromatography. The protein was extracted and concentrated by grinding under liquid nitrogen and ammonium sulfate fractionation. C-PC was purified by single step hydrophobic interaction chromatography. Purified phycocyanin showed absorbance maximum ( λ max) at 624 nm. The criterion of purity ( R) achieved was 4.52. Phycocyanin to phycoerythrin and phycocyanin to allophycocyanin purity ratio were 3.85 and 7.49, respectively. The purified protein showed a p I of 5.2 and has two subunits with molecular mass of 19 and 20 kDa each, corresponding to its highly reported α and β subunits. The subunits of phycocyanin were confirmed by their bilin fluorescence using zinc assisted florescence enhancement technique. Intact C-PC was of 125 kDa as determined by HPLC, suggested the (αβ) 3 subunit assembly. Results obtained by this method in terms of purity, recovery, process time, simplicity, and efficacy are much better than previous methodologies. Purified phycocyanin was further scrutinized for its antioxidant capacity and judged against five non-enzymatic antioxidants by FRAP assay.

Determination of Cyanobacterial Diversity during Algal Blooms in Daechung Reservoir, Korea, on the Basis of cpcBA Intergenic Spacer Region Analysis

The detection and prevention of cyanobacterial blooms are important issues in water quality management. As such, the diversity and community dynamics of cyanobacteria during cyanobacterial bloom in the Daechung Reservoir, Korea, were studied by analyzing the intergenic spacer (IGS) region between phycocyanin subunit genes cpcB and cpcA (cpcBA IGS). To amplify the cpcBA IGS from environmental samples, new PCR primers that could cover a wider range of cyanobacteria than previously known primers were designed. In the samples taken around the bloom peak (2 September 2003), seven groups of cpcBA IGS sequences were detected, and none of the amplified cpcBA IGSs was closely related to the cpcBA IGS from chloroplasts. Apart from the Microcystis-, Aphanizomenon (Anabaena)-, Pseudanabaena-, and Planktothrix (Oscillatoria)-like groups, the three other groups of cpcBA IGS sequences were only distantly related to previously reported sequences (<85% similarity to their closest relatives). The most prominent changes during the bloom were the gradual decrease and eventual disappearance of the Aphanizomenon (Anabaena)-like group before the bloom peak and the gradual increase and sudden disappearance of Planktothrix (Oscillatoria)-like groups right after the bloom peak. The community succession profile obtained based on the cpcBA IGS analysis was also supported by a PCR-denaturing gradient gel electrophoresis analysis of the 16S rRNA genes.