RESUMO
R-phycoerythrin (R-PE) is the most abundant, naturally occurring phycobiliproteins found in red algae. The spectroscopic and structural properties of phycobiliproteins exhibit unique absorption characteristics with two significant absorption maxima at 498 and 565 nm, indicating two different chromophores of R-PE, phycourobilin and phycoerythrobilin respectively. This study aimed to clarify how the stability of R-PE purified from F. lumbricalis was affected by different purification strategies. Crude extracts were compared to R-PE purified by i) microfiltration, ii) ultrafiltration, and iii) multi-step ammonium sulphate precipitation followed by dialysis. The stability of the different R-PE preparations was evaluated with respect to pH (2, 4, 6, 7, 8, 10 and 12) and temperature (20, 40, 60, 80 and 100 °C). The absorbance spectra indicated higher stability of phycourobilin as compared to phycoerythrobilin for heat and pH stability in the samples. All preparations of R-PE showed heat stability till 40 °C from the findings of color, concentration of R-PE and fluorescence emission. The crude extract showed stability from pH 6 to 8, whereas R-PE purified by ultrafiltration and multi-step ammonium sulphate precipitation were both stable from pH 4 to 8 and R-PE purified by microfiltration exhibited stability from pH 4 to 10 from the results of color, SDS-PAGE, and concentration of R-PE. At pH 2, the color changed to violet whereas a yellow color was observed at pH 12 in the samples along with the precipitation of the protein.
Assuntos
Ficoeritrina , Rodófitas , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Concentração de Íons de Hidrogênio , Rodófitas/química , Ultrafiltração/métodos , Estabilidade Proteica , Precipitação Química , Sulfato de Amônio/química , Temperatura Alta , TemperaturaRESUMO
Red phycoerythrin (R-PE) is a highly valuable protein found in an edible seaweed, Pyropia yezoensis. It is used extensively in biotechnological applications due to its strong fluorescence and stability in diverse environments. However, the current methods for extracting and purifying R-PE are costly and unsustainable. The aim of the present study was to enhance the financial viability of the process by improving the extraction and purification of R-PE from dried P. yezoensis and to further enhance R-PE value by incorporating it into a tandem dye for molecular biology applications. A combination of ultrafiltration, ion exchange chromatography, and gel filtration yielded concentrated (1 mg·mL-1) R-PE at 99% purity. Using purified PE and Cyanine5 (Cy5), an organic tandem dye, phycoerythrin-Cy5 (PE-Cy5), was subsequently established. In comparison to a commercially available tandem dye, PE-Cy5 exhibited 202.3% stronger fluorescence, rendering it suitable for imaging and analyzes that require high sensitivity, enhanced signal-to-noise ratio, broad dynamic range, or shorter exposure times to minimize potential damage to samples. The techno-economic analysis confirmed the financial feasibility of the innovative technique for the extraction and purification of R-PE and PE-Cy5 production.
Assuntos
Carbocianinas , Ficoeritrina , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Carbocianinas/química , Alga Marinha/química , Corantes Fluorescentes/química , Cromatografia por Troca Iônica/métodos , Cromatografia em Gel/métodos , Ultrafiltração/métodos , Rodófitas/química , Pigmentos Biológicos/isolamento & purificação , Pigmentos Biológicos/química , Algas Comestíveis , PorphyraRESUMO
Phycoerythrin (PE) is a pink/red-colored pigment found in rhodophytes, cryptophytes, and blue-green algae (cyanobacteria). The interest in PE is emerging from its role in delivering health benefits. Unfortunately, the current cyanobacterial-PE (C-PE) knowledge is still in the infant stage. It is essential to acquire a more comprehensive understanding of C-PE. This study aimed to review the C-PE structure, up and downstream processes of C-PE, application of C-PE, and strategies to enhance its stability and market value. In addition, this study also presented a strengths, weaknesses, opportunities, and threats (SWOT) analysis on C-PE. Cyanobacteria appeared to be the more promising PE producers compared to rhodophytes, cryptophytes, and macroalgae. Green/blue light is preferred to accumulate higher PE content in cyanobacteria. Currently, the prominent C-PE extraction method is repeated freezing-thawing. A combination of precipitation and chromatography approaches is proposed to obtain greater purity of C-PE. C-PE has been widely exploited in various fields, such as nutraceuticals, pharmaceuticals, therapeutics, cosmetics, biotechnology, food, and feed, owing to its bioactivities and fluorescent properties. This review provides insight into the state-of-art nature of C-PE and advances a step further in commercializing this prospective pigment.
Assuntos
Cianobactérias , Ficoeritrina , Humanos , Cromatografia , Cianobactérias/química , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Estudos Prospectivos , Rodófitas/químicaRESUMO
A single-step and rapid chromatographic method-based purification of Gracilaria corticata (J. Agardh) R-phycoerythrin (R-PE) was attained using polyacrylamide gel electrophoresis (PAGE) technique without affecting structural integrity. The purified R-PE had a characteristic UV-Vis spectrum with three absorbance maxima at 496, 535, and 565 nm, and fluorescence at 575 nm. R-PE was obtained with a purity index of 4.2 and a recovery yield of 44.3%. SDS-PAGE analysis exhibited three sub-units i.e., 18, 21, and 31 kDa, which corresponds to α, ß, and γ, respectively. This report's purification process was considered less time-consuming and could be efficiently applied to purify phycobiliproteins. The purified R-PE showed optimal stability up to 6 h at pH 7.0 when exposed to light (3000 lx), while the temperature at which the maximum stability was retained was at 20 °C. The cellular imaging property of R-PE was effectively implemented to evaluate its credentials without affecting the cell proliferation of Vero and Hep-2 cell lines with the higher IC50 concentrations in vitro. Under fluorescence microscopy and flow cytometry analysis, purified R-PE displayed the characteristic affinity towards cell imaging functions in preliminary in vitro studies.
Assuntos
Corantes Fluorescentes , Gracilaria/metabolismo , Imagem Óptica/métodos , Ficoeritrina , Animais , Chlorocebus aethiops , Corantes Fluorescentes/química , Corantes Fluorescentes/isolamento & purificação , Células Hep G2 , Humanos , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Células VeroRESUMO
OBJECTIVE: To investigate the effects of Phycoerythrin (PE) on the human ovarian cancer cell line SKOV-3 and its antitumor mechanisms from a transcriptional point of view. METHODS: SKOV-3 cells were exposed to different concentrations of phycoerythrin. The efficiency of this treatment was evaluated through cell growth inhibition, changes in cell morphology, apoptosis and intracellular ROS levels. High throughput sequencing (RNA-seq) was performed to screen Differentially Expressed Genes (DEGs), which was verified using RT-PCR and Western blotting. RESULTS: PE showed a significant inhibitory effect on the growth of SKOV-3 cells in a time- and dose-dependent manner. H&E staining, electron microscopy and flow cytometry revealed that PE induced apoptosis in SKOV-3 cells. Transcriptome analysis showed that 2963 genes were differentially expressed between untreated or PEtreated cells. GO and KEGG pathway analyses identified 16 classical pathways that were enriched. We verified 8 DEGs including, JNK, GADD45A, EDEM2, RAD23, UBQLN, CAPN1, XBP1, and OS9. These results were consistent with the results from transcriptional sequences. CONCLUSION: The inhibitory effect of PE on SKOV-3 cells was a result of interaction with multiple pathways and signaling molecules. Among these, the ROS/JNK/Bcl-2 signaling pathway, upregulation of JNK, GADD45A and RAD23 as well as downregulation of XBP1 and OS9 played a critical role in the PE -induced apoptosis in human ovarian cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Gracilaria/química , Ficoeritrina/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A565/A280) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K2HPO4 (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.
Assuntos
Ficoeritrina/isolamento & purificação , Porphyra/química , Sulfato de Amônio , Colina/química , Eletroforese em Gel de Poliacrilamida , Química Verde , Solventes , Espectrofotometria Ultravioleta , Ureia/química , ÁguaRESUMO
Porphyridium purpureum is a rich source for producing phycoerythrin (PE); however, the PE content is greatly affected by culture conditions. Researchers have aimed to optimize the cultivation of P. purpureum for accumulation of PE. When traditional optimized culture conditions were used to cultivate P. purpureum, high PE contents were not usually achieved. In this study, an induced cultivation pattern was applied to P. purpureum for PE biosynthesis (i.e., an incremental approach by altering temperatures, light intensities, and nitrate concentrations). Results revealed that the induced pattern greatly improved the PE biosynthesis. The optimized PE content of 229 mg/L was achieved on the 12th cultivation day, which was a maximum PE content within one cultivation period and accounted for approximately 3.05% of the dry biomass. The induced cultivation pattern was highly suitable for PE synthesis in P. purpureum, which provided an important reference value to the large-scale production of PE.
Assuntos
Biomassa , Luz , Ficoeritrina , Porphyridium/crescimento & desenvolvimento , Ficoeritrina/biossíntese , Ficoeritrina/química , Ficoeritrina/isolamento & purificaçãoRESUMO
In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.
Assuntos
Antioxidantes/química , Proteínas de Plantas/química , Rodófitas/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular , Humanos , Peróxido de Hidrogênio/toxicidade , Japão , Ficobilinas/química , Ficobilinas/isolamento & purificação , Ficobilinas/farmacologia , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Ficoeritrina/farmacologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologiaRESUMO
Aqueous two-phase extraction (ATPE) of R-Phycoerythrin (R-PE), a fluorescent and commercially valuable protein, was carried out in two parallel schemes. In scheme-1, ATPE alone was employed, and in scheme-2, process-integration was attempted, where primary extract was subjected to precipitation before standardization of process parameters of ATPE. Scheme-1 with polyethylene glycol (PEG)-3350/potassium phosphate system has resulted in very low R-PE yield (26% w/w) without much enrichment in purity (0.3). In scheme-2, PEG-1450/potassium phosphate system (pH 6, tie-line length 12.26% and lower volume-ratio) has resulted in higher R-PE purity (0.74) with 72% yield. R-PE containing PEG-rich (top) phase was subjected to ultrafiltration, to remove phase forming components, resulting in further increase in R-PE purity (1.1). Overall enrichment in R-PE purity of 11-fold with 57% (w/w) yield and removal of about 95% of total sugars was achieved in scheme-2. Intactness of R-PE after processing was confirmed by absorbance and emission spectrum analysis.
Assuntos
Ficoeritrina/isolamento & purificação , Rodófitas/química , Fosfatos/química , Polietilenoglicóis/química , Compostos de Potássio/química , UltrafiltraçãoRESUMO
Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6-8â¯Kâ¯MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5â¯h at a flow rate of 0.5â¯ml/min, while the lower channel was eluted with water at a flow rate of 0.05â¯ml/min after sample charge, and the column was rotated at 200â¯rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical "three peaks" spectrum with absorbance maximum at 497, 538 and 565â¯nm. The Native-PAGE showed one single protein band and 20â¯kDa (subunits α and ß) and 30â¯kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αß)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.
Assuntos
Centrifugação/métodos , Cromatografia/métodos , Gracilaria/química , Ficoeritrina/isolamento & purificação , Extratos Vegetais/química , Ficoeritrina/análise , Ficoeritrina/químicaRESUMO
A microfluidic device for pH gradient chromatofocusing is presented, which performs creation of a micro-column, pH gradient generation, and fraction collection in a single device. Using a sieve micro-valve, anion exchange particles were packed into a microchannel in order to realize a solid-phase absorption column. To fractionate proteins according to their isoelectric points, elution buffer solutions with a stepwise pH gradient were prepared in 16 parallel mixing reactors and flowed through the micro-column, wherein a protein mixture was previously loaded. The volume of the column is only 20 nL, hence it allows extremely low sample consumption and fast analysis compared with a conventional system. We demonstrated separation of two proteins, albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and R-Phycoerythrin (R-PE), by using a microcolumn of commercial charged polymeric particles (Source 15Q). The microfluidic device can be used as a rapid diagnostic tool to analyse crude mixtures of proteins or nucleic acids and determine adsorption/desorption characteristics of various biochemical products, which can be helpful for scientific fundamental understanding as well as instrumental in various industrial applications, especially in early stage screening and process development.
Assuntos
Cromatografia/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Proteínas/isolamento & purificação , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Ficoeritrina/isolamento & purificação , Proteínas/análise , Soroalbumina Bovina/isolamento & purificaçãoRESUMO
In the present study, blue light absorbing pigment protein phycoerythrin (PE) is purified up to molecular grade purity from marine Halomicronema sp. R31DM. The purification method is based on the use of non-ionic detergent Triton-X 100 in ammonium sulphate precipitation. The purified PE is characterized for its antioxidant activity in vitro and in vivo. PE is noted to show substantial in vitro antioxidant activity probed by various biochemical assays. The PE moderated rise in the intracellular-ROS (reactive oxygen species) in wild type Caenorhabditis elegans upon heat and oxidative stress. Further, the antioxidant asset of PE is noted an expedient in averting the ROS associated abnormalities, i.e. impaired physiological behaviour (health span) and aging in C. elegans. The structural attributes of PE contributing to its antioxidant virtue are analysed; the presence of ample residues having antioxidant activity and chromophore-PEB in PE are identified as a source of its antioxidant activity. Furthermore, the stability of PE is assessed under three physico-chemical stresses, temperature, pH and oxidative stress.
Assuntos
Antioxidantes/química , Caenorhabditis elegans/efeitos dos fármacos , Halobacteriaceae/química , Ficoeritrina/química , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Caenorhabditis elegans/crescimento & desenvolvimento , Temperatura Alta , Concentração de Íons de Hidrogênio , Estresse Oxidativo/efeitos dos fármacos , Ficoeritrina/isolamento & purificação , Ficoeritrina/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
In vitro antioxidant virtue and life-prolonging effect of phycoerythrin (PE; a pigment protein isolated from Phormidium sp. A09DM) have been revealed in our previous reports (Sonani et al. in Age 36:9717, 2014a; Sonani et al. in Process Biochem 49:1757-1766, 2014b). It has been hypothesized that the PE expands life span of Caenorhabditis elegans (bears large resemblance with human aging pathways) due to its antioxidant virtue. This hypothesis is tested in present study by checking the effect of PE on intracellular reactive oxygen species (ROS) generation and associated physiological deformities using mouse and human skin fibroblasts, C. elegans, and Drosophila melanogaster Oregon R + and by divulging PE's structural attributes responsible for its antioxidant asset. PE treatment displayed noteworthy decrease of 67, 48, and 77 % in ROS level in mouse fibroblast (3T3-L1), human fibroblast, and C. elegans N2, respectively, arisen under chemical-induced oxidative stress. PE treatment delayed the development of paraquat-induced Alzheimer phenotype by 14.5 % in C. elegans CL4176. Furthermore, PE improved the locomotion of D. melanogaster Oregon R + under oxidative stress with simultaneous up-regulation in super-oxide dismutase and catalase activities. The existence of 52 Glu + Asp + His + Thr residues (having metal ion sequestration capacity), 5 phycoerythrobilin chromophores (potential electron exchangers) in PE's primary structure, and significant hydrophobic patches on the surface of its α- and ß-subunits are supposed to collectively contribute in the antioxidant virtues of PE. Altogether, results support the hypothesis that it is the PE's antioxidant asset, which is responsible for its life-prolonging effect and thus could be exploited in the therapeutics of ROS-associated abnormalities including aging and neurodegeneration in eukaryotes.
Assuntos
Eucariotos/efeitos dos fármacos , Eucariotos/metabolismo , Espaço Intracelular/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ficoeritrina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Células 3T3-L1 , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Caenorhabditis elegans/efeitos dos fármacos , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Paraquat/toxicidade , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Ficoeritrina/metabolismo , Agregação Patológica de Proteínas , Superóxido Dismutase/metabolismoRESUMO
We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 µmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and ß-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and ß-subunits. In addition, the LRY sequence was found in the ß-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and ß-subunits of PE (rPEα and rPEß, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEß: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Ficobiliproteínas/farmacologia , Rodófitas/química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Hidrólise , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Ficobiliproteínas/química , Ficobiliproteínas/isolamento & purificação , Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Ficoeritrina/farmacologia , Hidrolisados de Proteína/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologiaRESUMO
B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and ß and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level.
Assuntos
Cromatografia por Troca Iônica , Ficoeritrina/isolamento & purificação , Porphyridium/química , Resinas de Troca Aniônica/química , Cromatografia por Troca Iônica/economia , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida , Ficoeritrina/química , UltracentrifugaçãoRESUMO
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 2.1â Å resolution at 100â K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4â Å, c = 116.6â 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 564â nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.
Assuntos
Cianobactérias/química , Ficoeritrina/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Sulfato de Amônio , Cristalização , Cristalografia por Raios X , Cianobactérias/genética , Cianobactérias/metabolismo , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Ficobilinas/química , Ficoeritrina/genética , Ficoeritrina/isolamento & purificação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis.
Assuntos
Ficoeritrina/isolamento & purificação , Rodófitas/química , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Ficoeritrina/química , Espectrofotometria/métodosRESUMO
A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and ß and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima.
Assuntos
Ficoeritrina/química , Ficoeritrina/isolamento & purificação , Extratos Vegetais/química , Rodófitas/química , Cromatografia por Troca Iônica/métodos , Ficoeritrina/análiseRESUMO
In the present study, microwave-assisted extraction was first employed to extract the phycobiliproteins of Porphyridium purpureum (Pp). Freeze-dried Pp cells were subjected to microwave-assisted extraction (MAE) to extract phycoerythin (PE), phycocyanin (PC), and allophycocyanin (APC). MAE combined reproducibility and high extraction yields and allowed a 180- to 1,080-fold reduction of the extraction time compared to a conventional soaking process. The maximal PE extraction yield was obtained after 10-s MAE at 40 °C, and PE was thermally damaged at temperatures higher than 40 °C. In contrast, a flash irradiation for 10 s at 100 °C was the best process to efficiently extract PC and APC, as it combined a high temperature necessary to extract them from the thylakoid membrane to a short exposure to thermal denaturation. The extraction order of the three phycobiliproteins was coherent with the structure of Pp phycobilisomes. Moreover, the absorption and fluorescence properties of MAE extracted phycobiliproteins were stable for several months after the microwave treatment. Scanning electron microscopy indicated that MAE at 100 °C induced major changes in the Pp cell morphology, including fusion of the exopolysaccharidic cell walls and cytoplasmic membranes of adjacent cells. As a conclusion, MAE is a fast and high yield process efficient to extract and pre-purify phycobiliproteins, even from microalgae containing a thick exopolysaccharidic cell wall.
Assuntos
Ficobiliproteínas/isolamento & purificação , Ficocianina/isolamento & purificação , Ficoeritrina/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Micro-Ondas , Ficobiliproteínas/química , Ficocianina/química , Ficoeritrina/química , Porphyridium/químicaRESUMO
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 â¼15.45 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.