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1.
Food Chem ; 306: 125615, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622833

RESUMO

Phycocyanin (PC), a plant-based protein with interesting biological activity, is rarely directly applied in the food industry because it has structural and functional limitations. This study combined ultra-high-pressure (UHP) treatment with glycation to improve PC functionality and explored resulting structural changes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, scanning electron microscopy, Fourier-transform infrared spectroscopy, circular dichroism, and UV-visible spectroscopy. The UHP treatment obviously improved the speed and degree of glycation and the composite-modified PC (CM-PC) showed high solubility and good emulsifying and foaming performance. Scanning electron microscopy images showed the CM-PC surface was loose and fluffy. Gel electrophoresis, Fourier-transform infrared spectroscopy, and circular dichroism results demonstrated that the content of α-helix decreased from 78.1% in PC to 26.6% in CM-PC, and hydroxyl groups were introduced. UV-visible spectroscopy showed that the mechanism of composite modification involved stretching of the PC and promotion of binding with sugars.


Assuntos
Ficocianina/química , Spirulina/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Emulsões/metabolismo , Glicosilação , Pressão , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier
2.
J Chem Phys ; 151(14): 144101, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615220

RESUMO

Phycobiliprotein is a light-harvesting complex containing linear tetrapyrrole bilin pigments that are responsible for absorption and funneling the sun's energy in cryptophytes algae. In particular, the protein structure determines relative positions and orientations of the pigments and thus controls energy transfer pathways. The present research reveals the impact of molecular vibrations (in the 850-2700 cm-1 region) on excitation energy transfer in phycobiliprotein. The analysis of the excitation energy transfer pathways indicates a possibility of the coherent mechanism of energy transfer (delocalization) in central dihydrobiliverdin pigments and incoherent vibration-assisted energy transfer to peripheral phycocyanobilin pigments at a sub-picosecond time scale. A computational approach that enables modeling the dynamics of the excitation energy transfer with the quantum master equation formalism employing Huang-Rhys factors to describe electronic-vibrational coupling has been developed. The computational methodology has been implemented in PyFREC software.


Assuntos
Transferência de Energia , Ficocianina/química , Biliverdina/análogos & derivados , Biliverdina/química , Criptófitas/química , Modelos Químicos , Ficobilinas/química , Teoria Quântica , Software , Vibração
3.
Molecules ; 24(15)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31374946

RESUMO

Arthrospira platensis (spirulina) is considered a source of natural molecules with nutritional and health benefits. As the different storage forms can affect the quantity and quality of bioactive ingredients, the aim of the present work was to evaluate the effects of freezing, oven-drying and freeze-drying on chemical composition of spirulina biomass. Total proteins, photosynthetic pigments and antioxidants, were analyzed and compared to respective quantities in fresh biomass. The frozen sample exhibited the highest content of phycocyanin-C, phenols, and ascorbic acid, also respect to the fresh biomass. The highest total flavonoid amount was in the freeze-dried biomass. HPLC-DAD analysis of phenolic acids revealed the presence of the isoflavone genistein, known for its therapeutic role, in all the spirulina samples. The phosphomolybdenum method (TAC) and DPPH scavenging activity were applied to determine the antioxidant activity of different samples. The highest DPPH scavenging activity was detected in fresh and freeze-dried biomass and it was positively related to carotenoid content. A positive correlation indicated that carotenoids, chlorophyll, ascorbic acid and all phenolic compounds were the major contributors to the TAC activity in spirulina biomass. The results highlighted a different functional value of spirulina biomass, depending on the processing methods used for its storage.


Assuntos
Antioxidantes/química , Liofilização/métodos , Fotossíntese , Spirulina/química , Ácido Ascórbico/química , Carotenoides/química , Clorofila/química , Dessecação/métodos , Flavonoides/química , Fenóis/química , Ficocianina/química , Pigmentação
4.
Int J Biol Macromol ; 135: 62-68, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31121226

RESUMO

Phycocyanin (PC) is the principal pigment protein in the light-harvesting antenna of cyanobacteria. Here the biochemical characterization and the 1.51 Šcrystal structure of PC from cyanobacterium Nostoc sp. WR13 (Nst-PC) is reported. The P63 crystal lattice is composed of the minimal biological entities of Nst-PC, the (αß)3 trimeric rings. The structure has been refined to R factor 11.5% (Rfree 15.4%) using anisotropic atomic B factors. A phylogenetic study shows that the α and ß chains of Nst-PC are significantly clustered in a distinct clade with Acaryochloris marina. The structure was examined to look for any significant differences between Nst-PC and PC from non-desert species. Only minor differences were found in the chromophore microenvironments. The tentative energy transfer pathways in Nst-PC were modeled based on simple structural considerations.


Assuntos
Modelos Moleculares , Estrutura Molecular , Nostoc/enzimologia , Ficocianina/química , Cristalografia por Raios X , Transferência de Energia , Conformação Molecular , Ficobiliproteínas/química , Ficobilissomas/química , Ficocianina/isolamento & purificação , Análise Espectral
5.
Int J Biol Macromol ; 132: 615-628, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30940588

RESUMO

In this work, a chitosan-modified nanofiber membrane was fabricated and used to examine the permeation characteristics of C-phycocyanin (CPC) obtained from Spirulina platensis. The effects of NaCl concentration (0.1-1.0 M), chitosan coupling pH (6-8), chitosan coupling concentration (0.1-3.0%), algal solution pH (6-8), algal mass concentration (0.1-1.0% dw/v), and membrane flux (4.08 × 10-2-2.04 × 10-1 mL/min·cm2) on the penetration performance of the membrane for CPC were investigated. The results show that the order of binding selectivity of the membrane for these proteins is contaminating proteins (TP) > allophycocyanin (APC) > CPC. TP and APC molecules were more easily adsorbed by the chitosan-modified membrane, and the CPC molecules most easily penetrated the membrane without being adsorbed, enhancing CPC purity. The purification factor and total mass flux were 3.3 fold and 66%, respectively, in a single step.


Assuntos
Quitosana/química , Cromatografia/métodos , Membranas Artificiais , Nanofibras/química , Ficocianina/isolamento & purificação , Concentração de Íons de Hidrogênio , Ficocianina/química , Cloreto de Sódio/química , Spirulina/química , Propriedades de Superfície
6.
Food Funct ; 10(4): 1816-1825, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30806395

RESUMO

Phycocyanin (PC), a water-soluble biliprotein, exhibits potent anti-colon cancer properties. However, its application in functional foods is limited by the poor stability and low bioavailability of PC. In this study, we successfully encapsulated PC by coaxial electrospinning. The colon targeted release of PC was achieved with retention of the antioxidant activity of PC. The PC-loaded electrospun fiber mat (EFM) obtained inhibited HCT116 cell growth in a dose-dependent and time-dependent manner. In particular, the PC-loaded EFM exerted its anti-cancer activity by blocking the cell cycle at the G0/G1 phase and inducing cell apoptosis involving the decrease of Bcl-2/Bax, activation of caspase 3 and release of cytochrome c. This study suggests that co-axial electrospinning is an efficient and effective way to deliver PC and improve its bioavailability; thus, it represents a promising approach for encapsulating functional ingredients for colon cancer prevention.


Assuntos
Antineoplásicos/química , Neoplasias do Colo/prevenção & controle , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Ficocianina/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/fisiopatologia , Composição de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/instrumentação , Células HCT116 , Humanos , Ficocianina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
7.
Biochim Biophys Acta Bioenerg ; 1860(4): 286-296, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30703363

RESUMO

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77 K. The study demonstrates that if complexes are stabilized by high phosphate (900 mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50 mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Ficobilinas/metabolismo , Ficobilissomas/metabolismo , Ficocianina/metabolismo , Proteínas de Bactérias/química , Ficobilinas/química , Ficobilissomas/química , Ficocianina/química , Espectrometria de Fluorescência
8.
Biotechnol Adv ; 37(3): 422-443, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30797095

RESUMO

Phycobiliproteins are a group of water soluble proteins with an associated chromophore, responsible for the light-harvesting in cyanobacteria. They are divided in four main types: phycoerythrin, phycocyanin, phycoerythrocyanin and allophycocyanin, and they are characterized according to their structure and light quality absorption. Phycobiliproteins from cyanobacteria have been described as potential bioactive compounds, and recognized as high-valued natural products for biotechnological applications. Moreover, phycobiliproteins have been associated to antioxidant, anticancer and anti-inflammatory capacities among others. Thus, in order to produce phycobiliproteins from cyanobacteria for industrial application, it is necessary to optimize the whole bioprocess, including the processing parameters (such as light, nitrogen and carbon source, pH, temperature and salinity) that affects the growth and phycobiliprotein accumulation, as well as the optimization of phycobiliproteins extraction and purification. The aim of this review is to give an overview of phycobiliproteins not only in terms of their chemistry, but also in terms of their biotechnological applicability and the advances and challenges in the production of such compounds.


Assuntos
Biotecnologia/tendências , Cianobactérias/química , Ficobiliproteínas/química , Ficobilinas/química , Ficobiliproteínas/biossíntese , Ficobiliproteínas/genética , Ficocianina/química , Ficoeritrina/química
9.
Int J Nanomedicine ; 14: 339-351, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30655667

RESUMO

Background: Hepatic ischemia/reperfusion-induced pancreatic islet injury (HI/RIPII) was an important pathophysiological phenomenon in clinics. In the present study, we observed the effects of phycocyanin on HI/RIPII. However, the half-life of phycocyanin was extremely short and limited its use in vivo. Materials and methods: In order to overcome this shortcoming, poly(ethylene glycol)-b-(poly(l-glutamic acid)-g-polyethylenimine) (PEG-b-(PG-g-PEI)) was synthesized and estimated as a nanocarrier for lengthening delivery of phycocyanin through the abdominal subcutaneous injection in rats. Phycocyanin (isoelectric point=4.3) was encapsulated with PEG-b-(PG-g-PEI) via electrostatic interactions at pH 7.4. Results: In vitro phycocyanin was fast and efficiently encapsulated and showing efficient loading and sustained release. In vivo the anti-HI/RIPII function of phycocyanin/PEG-b-(PG-g-PEI) complex was surveyed in rats using free phycocyanin as the controls, and the results showed that phycocyanin/PEG-b-(PG-g-PEI) complex reduced HI/RIPII property and enlarged islet functionality. Conclusion: These results suggested that PEG-b-(PG-g-PEI) might be treated as a potential phycocyanin nanocarrier.


Assuntos
Ilhotas Pancreáticas/lesões , Ficocianina/uso terapêutico , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Ácido Poliglutâmico/análogos & derivados , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Alanina Transaminase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/metabolismo , Metabolismo Basal , Glicemia/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Liberação Controlada de Fármacos , Insulina/sangue , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/patologia , Malondialdeído/metabolismo , Ficocianina/química , Ficocianina/farmacologia , Polietilenoimina/química , Ácido Poliglutâmico/química , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/sangue , Superóxido Dismutase/metabolismo
10.
Biochem Biophys Res Commun ; 509(2): 577-584, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30600183

RESUMO

Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250 kDa, i.e., 27 kDa green fluorescence protein, 105 kDa allophycocyanin, 220 kDa C-phycocyanin, and 250 kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440 kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.


Assuntos
Cristalização/métodos , Decapodiformes/química , Hemocianinas/química , Animais , Proteínas de Fluorescência Verde/química , Modelos Moleculares , Ficocianina/química , Ficoeritrina/química , Porosidade
11.
J Photochem Photobiol B ; 190: 110-117, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30508759

RESUMO

The attribution of quantum beats observed in the time-resolved spectroscopy of photosynthetic light-harvesting antennae to nontrivial quantum coherences has sparked a flurry of research activity beginning a decade ago. Even though investigations into the functional aspects of photosynthetic light-harvesting were supported by X-ray crystal structures, the non-covalent interactions between pigments and their local protein environment that drive such function has yet to be comprehensively explored. Using symmetry-adapted perturbation theory (SAPT), we have comprehensively determined the magnitude and compositions of these non-covalent interactions involving light-harvesting chromophores in two quintessential photosynthetic pigment-protein complexes - peridinin chlorophyll-a protein (PCP) from dinoflagellate Amphidinium carterae and phycocyanin 645 (PC645) from cryptophyte Chroomonas mesostigmatica. In PCP, the chlorophylls are dispersion-bound to the peridinins, which in turn are electrostatically anchored to the protein scaffold via their polar terminal rings. This might be an evolutionary design principle in which the relative orientation of the carotenoids towards the aqueous environment determines the arrangement of the other chromophores in carotenoid-based antennas. On the other hand, electrostatics dominate the non-covalent interactions in PC645. Our ab initio simulations also suggest full protonation of the PC645 chromophores in physiological conditions, and that changes to their protonation states result in their participation as switches between folded and unfolded conformations.


Assuntos
Criptófitas/química , Dinoflagelados/química , Complexos de Proteínas Captadores de Luz/química , Modelos Teóricos , Carotenoides/química , Clorofila , Conformação Molecular , Ficocianina/química , Proteínas/metabolismo , Proteínas de Protozoários/química , Eletricidade Estática
12.
Biochim Biophys Acta Mol Cell Res ; 1866(2): 277-284, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30471307

RESUMO

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700-770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650-700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.


Assuntos
Bilirrubina/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Proteínas de Bactérias/metabolismo , Bilirrubina/metabolismo , Biliverdina/química , Biliverdina/metabolismo , Cianobactérias/metabolismo , Escherichia coli/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Humanos , Luz , Microscopia de Fluorescência , Ficobilissomas/metabolismo , Ficocianina/química , Ficocianina/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos
13.
Colloids Surf B Biointerfaces ; 175: 221-230, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30537618

RESUMO

C-Phycocyanin pigment was purified from a native cyanobacterial strain using a novel consecutive multi-step procedure and utilized for the first time for the green synthesis of phycocyanin-zinc oxide nanorods (PHY-ZnO NRs) by a simple, low-cost and eco-friendly approach. The PHY-ZnO NRs were characterized using UV-vis spectroscopy, X-ray diffraction (XRD), zeta potential measurement, FTIR, SEM, TEM, differential scanning calorimetry (DSC), thermogravimetric (TGA), and EDX spectroscopy analysis. The UV-vis spectra showed an absorption band at 364 nm which is characteristic of ZnO nanoparticles (ZnONPs). The rod-shaped PHY-ZnO NRs observed in the TEM and SEM images had an average diameter size of 33 nm, which was in good agreement with the size calculated by XRD. The elemental analysis of PHY-ZnO NRs composition showed that three emission peaks of zinc metal and one emission peak of oxygen comprised 33.88% and 42.50%, respectively. The thermogram of PHY-ZnO NRs sample exhibited the weight loss of biosynthesized nanoparticles registered to be 3%, emphasizing the purity and heat stability of zinc oxide nanorods coated with phycocyanin pigment-protein. MTT assay indicated that PHY-ZnO NRs had a less cytotoxicity on fibroblast L929 compared to the ZnONRs-treated cells. A remarkable increase in ROS level was measured in cells treated with ZnO at final concentrations of 100, 200 and 500 µg/ml (78 ± 7, 99 ± 8 and 116 ± 11, respectively). When it comes to PHY-ZnO NRs, a protective effect for phycocyanin was detected which declined the level of ROS content as confirmed by fluorescent microscopy. The distinctive features of phycocyanin for surface functionalization of ZnO nanoparticles deserve to be deemed as a nano-drug candidate for further researches.


Assuntos
Nanopartículas Metálicas/química , Nanotubos/química , Ficocianina/química , Óxido de Zinco/química , Administração Oral , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Masculino , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanotubos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
14.
J Chromatogr A ; 1587: 119-128, 2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30579643

RESUMO

Monolithic adsorbers with anion exchange (AEX) properties have been 3D printed in an easy one-step process, i.e. not requiring post-functionalization to introduce the AEX ligands. The adsorber, 3D printed using a commercial digital light processing (DLP) printer, was obtained by copolymerisation of a bifunctional monomer bearing a positively charged quaternary amine as well as an acrylate group, with the biocompatible crosslinker polyethylene glycol diacrylate (PEGDA). To increase the surface area, polyethylene glycol was introduced into the material formulation as pore forming agent. The influence of photoinitiator (Omnirad 819) and photoabsorber (Reactive Orange 16, RO16) concentration was investigated in order to optimize printing resolution, allowing to reliably 3D print features as small as 200 µm and of highly complex Schoen Gyroids. Protein binding was measured on AEX adsorbers with a range of ligand densities (0.00, 2.03, 2.60 and 3.18 mmol/mL) using bovine serum albumin (BSA) and c-phycocyanin (CPC) as model proteins. The highest equilibrium binding capacity was found for the material presenting the lowest ligand density analysed (2.03 mmol/mL), adsorbing 73.7 ± 5.9 mg/mL and 38.0 ± 2.2 mg/mL of BSA and CPC, respectively. This novel 3D printed material displayed binding capacities in par or even higher than commercially available chromatographic resins. We expect that the herein presented approach of using bifunctional monomers, bearing commonly used chromatography ligands, will help overcome the material limitations currently refraining 3D printing applications in separation sciences.


Assuntos
Impressão Tridimensional , Acrilatos/química , Adsorção , Animais , Ânions , Bovinos , Cromatografia por Troca Iônica/métodos , Troca Iônica , Ficocianina/química , Polietilenoglicóis/química , Porosidade , Compostos de Amônio Quaternário/química , Soroalbumina Bovina/química
15.
Int J Biol Macromol ; 126: 685-696, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30557646

RESUMO

Phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates showing special spectral characteristics were prepared from the partly dissociated products of phycobilisomes from Polysiphonia urceolata. The absorption difference spectra between the aggregates and phycoerythrins showing normal spectral characteristics show peaks at 583 nm. The fluorescence emission difference spectra between the phycoerythrin aggregates and normal phycoerythrins show peaks at 602 nm. The special spectral characteristics of the PE aggregates disappeared when the PE aggregates were dissociated in deionized water. The intact phycobilisomes show similar characteristics as phycoerythrin-phycocyanin aggregates. When the peak values at 583 nm in absorption difference spectra were compared with those at 615 nm, it can be found that the amount of the 583 nm chromophores in the phycoerythrin-phycocyanin aggregates and intact phycobilisomes is similar to the amount of PCB chromophores in phycocyanins. It can be concluded that the 583 nm chromophores are at the interface between phycoerythrins and phycocyanins in phycobilisomes and transfer the light energy absorbed by other chromophores in phycoerythrins to the PCB chromophores in phycocyanins. A rod linker polypeptide with molecular weight of 40 kDa was found in the phycoerythrin-phycocyanin aggregates and phycoerythrin aggregates, and it is believed to play roles in the spectral red shift of the aggregates.


Assuntos
Organismos Aquáticos/química , Ficobilissomas/química , Ficocianina/química , Ficoeritrina/química , Rodófitas/química , Centrifugação com Gradiente de Concentração , Ficobiliproteínas/química , Espectrometria de Fluorescência
16.
J Biosci Bioeng ; 126(6): 778-782, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30401453

RESUMO

Fusion protein of streptavidin and allophycocyanin holo-α subunit (ApcA) is fluorescent and is able to bind biotin. This fusion protein (SLA) can be used as fluorescent label in immunofluorescence assay. In this study, one or two repeats of ApcA were fused to the protein SLA, with the aim to improve its brightness. The fusion proteins SLA2 (two repeats of ApcA) and SLA3 (three repeats of ApcA), together with lyase (cpcS) and phycoerythrobilin synthesizing enzymes (Ho1 and PebS), were co-expressed in Escherichia coli. These fusion proteins were purified by affinity chromatography. While SLA2 and SLA3 shared similar absorbance spectra, fluorescence spectra and biotin-binding activities with SLA, their brightness were much higher than that of SLA. When used as fluorescent labels in immunofluorescence assay, SLA2 and SLA3 showed higher detection sensitivity than SLA. These results suggested that SLA2 and SLA3 were the preferable fluorescent labels in immunofluorescence assays.


Assuntos
Escherichia coli/metabolismo , Imunofluorescência/métodos , Corantes Fluorescentes/metabolismo , Ficocianina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Corantes Fluorescentes/química , Liases/genética , Liases/metabolismo , Ficobilinas/química , Ficobilinas/metabolismo , Ficocianina/química , Ficoeritrina/química , Ficoeritrina/metabolismo , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Estreptavidina/química , Estreptavidina/metabolismo
17.
Biotechnol Prog ; 34(5): 1261-1268, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30281951

RESUMO

C-phycocyanin (C-PC) is a natural blue dye, and depending on its purity, which is measured by the ratio between the absorbance of the chromophore (A620 ) and the absorbance of the proteins (A280 ), it can be used in food (purity > 0.7), cosmetics (purity > 1.5), and therapeutic treatments (purity > 4.0). Several physical, chemical, and enzymatic methods of extraction are reported, however, few are able to extract C-PC with purity above 0.7. An innovative method of C-PC extraction with food grade purity from wet Spirulina platensis biomass is proposed. The cells were pretreated with ethylenediaminetetraacetic acid and subsequent C-PC extraction was performed with tris-(hydroxymethyl) aminomethane-SO4 buffer. C-PC was released after 12 h of cell pretreatment. Six variables of the extraction process were evaluated. The extraction temperature significantly influenced C-PC extraction yield and purity. In the best condition of cell pretreatment and extraction, C-PC with purity of 1.0 and extraction yield of 129.0 mg/g could be obtained to be used as a food dye without any purification process. Lastly, an ultrafiltration process was integrated and C-PC was concentrated 8.8-fold, resulting in purity of 1.6 and recovery of 93.4%.


Assuntos
Ácido Edético/química , Ficocianina/química , Ficocianina/isolamento & purificação
18.
Bioresour Technol ; 270: 320-327, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30241065

RESUMO

An aqueous two-phase system (ATPS) with ionic liquids (ILs) was used for the isolate of C-phycocyanin (CPC) from Spirulina platensis microalga. Various imidazolium ILs and potassium salts were studied. The effect of ILs-ATPS on the extraction efficiency of CPC was also studied. The experimental parameters like pH, loading volume, algae concentration, temperature, and alkyl chain length of IL were well-covered in this report. The experimental results showed that the extraction efficiency, the partition coefficient, and the separation factor for CPC were 99%, 36.6, and 5.8, respectively, for an optimal pH value of 7 and a temperature of 308 K. The order of extraction efficiency for CPC using IL-ATPS was: 1-octyl-3-methylimidazolium bromide (C8MIM-Br) > 1-hexyl-3-methylimidazolium bromide (C6MIM-Br) > 1-butyl-3-methylimidazolium bromide (C4MIM-Br). The isolation process followed the pseudo second-order kinetic model and the thermodynamic results were obviously spontaneous.


Assuntos
Microalgas/metabolismo , Ficocianina/metabolismo , Spirulina/metabolismo , Boratos/química , Imidazóis/química , Líquidos Iônicos/química , Microalgas/química , Ficocianina/química , Spirulina/química , Temperatura Ambiente , Termodinâmica
19.
J Agric Food Chem ; 66(41): 10921-10929, 2018 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-30253646

RESUMO

As a type of functional food additive, phycocyanin is shown to have a potential antineoplastic property. However, its underlying anticancer mechanism in melanoma cells remains unknown. We previously reported a 35S in vivo/vitro labeling analysis for dynamic proteomic (SiLAD) technology. It could exclusively detect protein synthesis rates via pulse labeling of newly expressed proteins by 35S, providing a high time-resolution method for analysis of protein variations. In the present study, we performed a time course analysis in A375 melanoma cells after phycocyanin treatment using SiLAD. Protein expression velocities were specifically visualized and their regulation modes were dynamically traced. Strikingly, novel protein synthesis patterns were discovered in the early phase of phycocyanin treatment, suggesting a possible mechanism of phycocyanin regulation. Furthermore, network analysis and phenotype experiments demonstrated that GRB2-ERK1/2 pathway was involved in phycocyanin-mediated regulation process and responsible for the proliferation suppression of melanoma cell, which could be a therapeutic target for malignant melanoma.


Assuntos
Antineoplásicos/química , Aditivos Alimentares/química , Melanoma/tratamento farmacológico , Ficocianina/química , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Aditivos Alimentares/metabolismo , Aditivos Alimentares/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ficocianina/metabolismo , Ficocianina/uso terapêutico , Proteínas/metabolismo , Proteômica/métodos
20.
J Proteome Res ; 17(11): 3628-3643, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30216071

RESUMO

The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N2 fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO2 uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.


Assuntos
Proteínas de Bactérias/química , Cyanothece/química , Complexos Multiproteicos/química , Ficocianina/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Dióxido de Carbono/metabolismo , Cromatografia em Gel , Ciclo do Ácido Cítrico/fisiologia , Biologia Computacional , Cyanothece/metabolismo , Glicólise/fisiologia , Espectrometria de Massas , Complexos Multiproteicos/metabolismo , Nitrogênio/metabolismo , Fixação de Nitrogênio/fisiologia , Oxigênio/metabolismo , Fosforilação , Fotossíntese/fisiologia , Ficocianina/química , Mapeamento de Interação de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteoma/isolamento & purificação , Proteoma/metabolismo , Proteômica/métodos
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