Red/green cyanobacteriochromes acquire isomerization from phycocyanobilin to phycoviolobilin.

Cyanobacteriochromes (CBCRs) are unique cyanobacteria-specific photoreceptors that share a distant relation with phytochromes. Most CBCRs contain conserved cysteine residues known as canonical Cys, while some CBCRs have additional cysteine residues called second Cys within the DXCF motif, leading to their classification as DXCF CBCRs. They typically undergo a process where they incorporate phycocyanobilin (PCB) and subsequently isomerize it to phycoviolobilin (PVB). Conversely, CBCRs with conserved Trp residues and without the second Cys are called extended red/green (XRG) CBCRs. Typical XRG CBCRs bind PCB without undergoing PCB-to-PVB isomerization, displaying red/green reversible photoconversion, and there are also atypical CBCRs that exhibit diverse photoconversions. We discovered novel XRG CBCRs with Cys residue instead of the conserved Trp residue. These novel XRG CBCRs exhibited the ability to isomerize PCB to PVB, displaying green/teal reversible photoconversion. Through sequence- and structure-based comparisons coupled with mutagenesis experiments, we identified three amino acid residues, including the Cys residue, crucial for facilitating PCB-to-PVB isomerization. This research expands our understanding of the diversity of XRG CBCRs, highlighting the remarkable molecular plasticity of CBCRs.

Optimizing Phycocyanin Extraction from Cyanobacterial Biomass: A Comparative Study of Freeze-Thaw Cycling with Various Solvents.

Cyanobacterial phycocyanin pigment is widely utilized for its properties in various industries, including food, cosmetics, and pharmaceuticals. Despite its potential, challenges exist, such as extraction methods impacting yield, stability, and purity. This study investigates the impact of the number of freeze-thaw (FT) cycles on the extraction of phycocyanin from the wet biomass of four cyanobacteria species (Arthrospira platensis, Chlorogloeopsis fritschii, Phormidium sp., and Synechocystis sp.), along with the impact of five extraction solutions (Tris-HCl buffer, phosphate buffer, CaCl2, deionized water, and tap water) at various pH values. Synechocystis sp. exhibited the highest phycocyanin content among the studied species. For A. platensis, Tris-HCl buffer yielded maximum phycocyanin concentration from the first FT cycle, while phosphate buffer provided satisfactory results from the second cycle. Similarly, Tris-HCl buffer showed promising results for C. fritschii (68.5% of the maximum from the first cycle), with the highest concentration (~12% w/w) achieved during the seventh cycle, using phosphate buffer. Phormidium sp. yielded the maximum pigment concentration from the first cycle using tap water. Among species-specific optimal extraction solutions, Tris-HCl buffer demonstrated sufficient extraction efficacy for all species, from the first cycle. This study represents an initial step toward establishing a universal extraction method for phycocyanin from diverse cyanobacteria species.

Teal-light absorbing cyanobacterial phytochrome superfamily provides insights into the diverse mechanisms of spectral tuning and facilitates the engineering of photoreceptors for optogenetic tools.

Cyanobacteriochromes (CBCRs) are distinctive tetrapyrrole (bilin)-binding photoreceptors exclusively found in cyanobacteria. Unlike canonical phytochromes, CBCRs require only a GAF (cGMP-phosphodiesterase/adenylate cyclase/FhlA) domain for autolyase activity to form a bilin adduct via a Cys residue and cis-trans photoisomerization. Apart from the canonical Cys, which attaches covalently to C31 in the A-ring of the bilin, some GAF domains of CBCRs contain a second-Cys in the Asp-Xaa-Cys-Phe (DXCF) motif, responsible for isomerization of phycocyanobilin (PCB) to phycoviolobilin (PVB) and/or for the formation of a reversible 2nd thioether linkage to the C10. Unlike green/teal-absorbing GAF proteins lacking ligation activity, the second-Cys in another teal-absorbing lineage (DXCF blue/teal group) exhibits both isomerization and ligation activity due to the presence of the Tyr instead of His next to the canonical Cys. Herein, we discovered an atypical CBCR GAF protein, Tpl7205g1, belonging to the DXCF blue/teal group, but having His instead of Tyr next to the first-Cys. Consistent with its subfamily, the second-Cys of Tpl7205g1 did not form a thioether linkage at C10 of PCB, showing only isomerization activity. Instead of forming 2nd thioether linkage, this novel GAF protein exhibits a pH-dependent photocycle between protonated 15Z and deprotonated 15E. Site-directed mutagenesis to the GAF scaffolds revealed its combined characteristics, including properties of teal-DXCF CBCRs and red/green-absorbing CBCRs (XRG CBCRs), suggesting itself as the evolutionary bridge between the two CBCR groups. Our study thus sheds light on the expanded spectral tuning characteristics of teal-light absorbing CBCRs and enhances feasibility of engineering these photoreceptors.

Phycocyanin-phlorotannin complexes improve the structure and functional properties of yogurt.

Adding natural bioactive ingredients to yogurt can improve the nutritional and physiological benefits. In this study, we used ultrasonic-assisted phlorotannin from Ascophyllum nodosum (A. nodosum) modified phycocyanin (PC) to form a complex (UPP) to produce a fortified fermented yogurt. The effects of PC and UPP on the structure, stability, and function of fermented yogurt within 7 days were assessed using physicochemical properties, texture analysis, rheological testing, 16S rDNA sequencing analysis, and lipidomics analysis. Molecular docking indicated that PC might bind to phlorotannin via ARG-77, ARG-84, LEU-120, ALA-81, CYS-82, and ASP-85 sites.When the mass ratio of the complex is 1:1, the ability of UPP1:1 to remove DPPH· scavenging ability in an acid environment increased by about 50 %. UPP1:1 with more acid stability changed the microstructure of the yogurt, enhanced the stability of the yogurt, improved the antioxidant properties, and inhibited the growth of harmful bacteria within 7 days. This work encouraged the extraction and use of phlorotannin from edible brown algae and offered a straightforward method for making yogurt supplemented with PC.

Construction of a pectin/sodium alginate composite hydrogel delivery system for improving the bioaccessibility of phycocyanin.

In this study, different concentrations of sodium alginate were compounded with pectin and phycocyanin to co-prepare composite hydrogel spheres (HP-PC-SA 0.2 %, 0.6 %, 1.0 %, 1.4 %) to evaluate the potential of the composite hydrogel spheres for the application as phycocyanin delivery carriers. The hydrogel spheres' physicochemical properties and bioaccessibility were assessed through scanning electron microscopy, textural analysis, drug-carrying properties evaluation, and in vitro and in vivo controlled release analysis in the gastrointestinal environment. Results indicated that higher sodium alginate concentrations led to smaller pore sizes and denser networks on the surface of hydrogel spheres. The textural properties of hydrogel spheres improved, and their water-holding capacity increased from 93.01 % to 97.97 %. The HP-PC-SA (1.0 %) formulation achieved the highest encapsulation rate and drug loading capacity, at 96.87 % and 6.22 %, respectively. Within the gastrointestinal tract, the composite hydrogel's structure significantly enhanced and protected the phycocyanin's digestibility, achieving a bioaccessibility of up to 88.03 %. In conclusion, our findings offer new insights into improving functionality and the effective use of phycocyanin via pectin-based hydrogel spheres.

Making a living off the rainbow's edge: How phycobilisomes adapt structurally to absorb far-red light.

Cyanobacteria harvest light by using architecturally complex, soluble, light-harvesting complexes known as phycobilisomes (PBSs). PBS diversity includes specialized subunit paralogs that are tuned to specific regions of the light spectrum; some cyanobacterial lineages can even absorb far-red light. In a recent issue of the Journal of Biological Chemistry, Gisriel et al. reported the cryo-electron microscopic structure of a far-red PBS core, showing how bilin binding in the α-subunits of allophycocyanin paralogs can modify the bilin-binding site to red shift the absorbance spectrum. This work helps explain how cyanobacteria can grow in environments where most of the visible light has been filtered out.

Characterization of emulgels formulated with phycocyanin and diutan gum as a novel approach for biocompatible delivery systems.

Phycocyanin (PC), a protein derived from algae, is non-toxic and biocompatible. Due to its environmental and sustainable properties, it has been studied as an alternative stabilizer for food emulsions. In this sense, the main objective of this work is to evaluate the effectiveness of PC and its use in combination with diutan gum (DG), a biological macromolecule, to prepare emulgels formulated with avocado oil. Z-potential measurements show that the optimum pH for working with PC is 2.5. Furthermore, the system exhibited a structured interface at this pH. The surface tension did not decrease further above 1.5 wt% PC. Interestingly, emulsions formulated with >1.5 wt% PC showed recoalescence immediately after preparation. Although 1.5 wt% had the smallest droplet size, this emulsion underwent creaming due to the low viscosity of the system. DG was used in combination with PC to increase viscosity and reduce creaming. As little as 0.1 wt% DG was sufficient to form an emulgel when incorporated into the previous emulsion, which exhibited pseudoplastic behaviour and viscoelastic properties with very low creaming rates. However, the use of PC in combination with DG resulted in a non-aggregated and stable emulgel with 1.5 wt% PC and 0.1 wt% DG.

A rapid one-step affinity purification of C-phycocyanin from Spirulina platensis.

The high-purity phycocyanin has a high commercial value. Most current purification methods of C-phycocyanin involve multiple steps, which are complicated and time-consuming. To solve the problem, this research was studied, and an efficient affinity chromatography purification for C-phycocyanin from Spirulina platensis was developed. Through molecular docking simulation, virtual screening of ligands was performed, and ursolic acid was identified as the specific affinity ligand, which coupled to Affi-Gel 102 gel via 1-ethyl (3-dimethylaminopropyl)-3-carbodiimide, hydrochloride as coupling agent. With this customized and synthesized resin, a high-efficiency one-step purification procedure for C-phycocyanin was developed and optimized, the purity was determined to be 4.53, and the yield was 69 %. This one-step purification protocol provides a new approach for purifying other phycobilin proteins.

Collagen/chitosan/genipin hydrogel loaded with phycocyanin nanoparticles and ND-336 for diabetic wound healing.

Diabetic wound healing remains a healthcare challenge due to the overexpression of matrix metalloproteinase-9 (MMP-9) and the imbalance between angiogenic factors and vascular inhibitory factors. In this study, we developed a nanocomposite injectable collagen/chitosan hydrogel for the treatment of delayed diabetic wound healing, which can promote cell migration to the wound site (through the addition of phycocyanin) and reduce the expression of MMP-9 (through the use of ND-336) to improve the therapeutic effect of diabetic wound healing. Furthermore, different weight ratios of collagen and chitosan hydrogels were prepared to select the hydrogel with proper mechanical properties. In vitro experiments confirmed that all hydrogels have favorable biocompatibility and hemocompatibility. Notably, Gel 2, with a weight ratio of collagen and chitosan at 25:75, was found to have an excellent capability to facilitate cell migration and in vivo studies further proved that Gel 2 nanocomposite hydrogel had the best ability to improve diabetic wound healing by promoting cell migration and decreasing MMP-9 expression. The collagen/chitosan/genipin hydrogel loaded phycocyanin and ND-336 can be harnessed for non-toxic and efficient treatment of wound healing management of diabetes.

Assembly of CpcL-phycobilisomes.

CpcL-phycobilisomes (CpcL-PBSs) are a reduced type of phycobilisome (PBS) found in several cyanobacteria. They lack the traditional PBS terminal energy emitters, but still show the characteristic red-shifted fluorescence at ~670 nm. We established a method of assembling in vitro a rod-membrane linker protein, CpcL, with phycocyanin, generating complexes with the red-shifted spectral features of CpcL-PBSs. The red-shift arises from the interaction of a conserved key glutamine, Q57 of CpcL in Synechocystis sp. PCC 6803, with a single phycocyanobilin chromophore of trimeric phycocyanin at one of the three ß82-sites. This chromophore is the terminal energy acceptor of CpcL-PBSs and donor to the photosystem(s). This mechanism also operates in PBSs from Acaryochloris marina MBIC11017. We then generated multichromic complexes harvesting light over nearly the complete visible range via the replacement of phycocyanobilin chromophores at sites α84 and ß153 of phycocyanins by phycoerythrobilin and/or phycourobilin. The results demonstrate the rational design of biliprotein-based light-harvesting elements by engineering CpcL and phycocyanins, which broadens the light-harvesting range and accordingly improves the light-harvesting capacity and may be potentially applied in solar energy harvesting.

Hepatoprotective Effect Assessment of C-Phycocyanin on Hepatocellular Carcinoma Rat Model by Using Photoacoustic Spectroscopy.

Hepatocellular carcinoma (HCC) is the most common primary neoplasia of the liver with elevated mortality. Experimental treatment with antioxidants has a beneficial effect on the experimental models of HCC. Arthrospira maxima (spirulina) and its phycocyanin have antitumoral action on different tumoral cells. However, it is unknown whether phycocyanin is the responsible molecule for the antitumoral effect on HCC. Photoacoustic spectroscopy (PAS) stands out among other spectroscopy techniques for its versatility of samples analyzed. This technique makes it possible to obtain the optical absorption spectrum of solid or liquid, dark or transparent samples. Previous studies report that assessing liver damage in rats produced by the modified resistant hepatocyte model (MRHM) is possible by analyzing their blood optical absorption spectrum. This study aimed to investigate, by PAS, the effect of phycocyanin obtained from spirulina on hepatic dysfunction. The optical absorption spectra analysis of the rat blood indicates the damage level induced by the MRHM group, being in concordance with the carried out biological conventional studies results, indicating an increase in the activity of hepatic enzymes, oxidative stress, Bax/Bcl2 ratio, cdk2, and AKT2 expression results, with a reduction in p53 expression. Also, PAS results suggest that phycocyanin decreases induced damage, due to the prevention of the Bax, AKT2, and p53 altered expression and the tumor progression in a HCC rat model.

Spectral and conformational characteristics of phycocyanin associated with changes of medium pH.

C-phycocyanin (C-PC) is the main component of water-soluble light-harvesting complexes (phycobilisomes, PBS) of cyanobacteria. PBS are involved in the absorption of quantum energy and the transfer of electronic excitation energy to the photosystems. A specific environment of C-PC chromophoric groups is provided by the protein matrix structure including protein-protein contacts between different subunits. Registration of C-PC spectral characteristics and the fluorescence anisotropy decay have revealed a significant pH influence on the chromophore microenvironment: at pH 5.0, a chromophore is more significantly interacts with the solvent, whereas at pH 9.0 the chromophore microenvironment becomes more viscous. Conformations of chromophores and the C-PC protein matrix have been studied by Raman and infrared spectroscopy. A decrease in the medium pH results in changes in the secondary structure either the C-PC apoproteins and chromophores, the last one adopts a more folded conformation.

Effects of chitosan molecular weight and mass ratio with natural blue phycocyanin on physiochemical and structural stability of protein.

Phycocyanin (PC), an algae-extracted colorant, has extensive applications for its water-solubility and fresh blue shade. When PC is added to acidified media, dispersions are prone to aggregate and decolorize into cloudy systems. For palliating this matter, chitosan with high, medium, and low molecular weights (HMC, MMC, and LMC) were adopted in PC dispersions, and their protective effects were compared based on physiochemical stabilities. The optimal mass ratio between chitosan and PC was identified as 1:5 based on preliminary evaluations and was supported by the higher ζ-potential (31.0-32.1 mV), lower turbidity (39.6-43.6 NTU), and polyacrylamide gel electrophoresis results. Through interfacial and antioxidant capacity analyses, LMC was found to display a higher affinity to PC, which was also confirmed by SEM images and the maximum increase in transition temperature of their complex (155.70 °C) in DSC measurements. The mechanism of electrostatic interaction reinforced by hydrophobic effects and hydrogen bonding was elucidated by FT-IR and Raman spectroscopy. Further comprehensive stability evaluations revealed that, without light exposure, LMC kept PC from internal secondary structure to external blueness luster to the maximum extent. While with light exposure, LMC was not so flexible as HMC, to protect chromophores from attack of free radicals.

Cooperative antioxidative defense of the blue-green alga Arthrospira (Spirulina) platensis under oxidative stress imposed by exogenous application of hydrogen peroxide.

Hydrogen peroxide (H2O2) is an environmentally-safe algaecide used to control harmful algal blooms and as a disinfectant in various domestic and industrial applications. It is produced naturally in sunny-water or as a by-product during growth, and metabolism of photosynthetic organisms. To assess the impact of H2O2 on Arthrospira platensis, several biochemical components, and antioxidant enzymes were analysed. The growth and biomass of A. platensis were decreased under the effect of H2O2. Whereas, the concentration up to 40 µM H2O2 non-significantly induced (at P < 0.05) the Chl a, C-phycocyanin (C-PC), total phycobiliprotein (PBP), and the radical scavenging activity of A. platensis. The half-maximal effective concentrations (EC50) for H2O2 were 57, 65, and 74 µM H2O2 with regards to the biomass yield, Chl a, and C-PC content, respectively. While, the total soluble protein, and soluble carbohydrates contents were significantly induced. However, the higher concentrations (60 and 80 µM) were lethal to these components, in parallel to the initiation of the lipid peroxidation process. Surprisingly, the carotenoids content was non-significantly increased by H2O2. Despite the relative consistency of catalase (CAT), the activities of superoxide dismutase (SOD) and peroxidase (POD) enzymes were boosted by all of the tested concentrations of H2O2. The relative transcript abundance of selected regulatory genes was also investigated. Except for the highest dose (80 µM), the tested concentrations had almost inhibitory effect on the relative transcripts of heat shock protein (HSP90), glutamate synthase (GOGAT), delta-9 desaturase (desC), iron-superoxide dismutase (FeSOD) and the Rubisco (the large subunit, rbcL) genes. The results demonstrated the importance of the non-enzymatic and enzymatic antioxidants for the cumulative tolerance of A. platensis.

Interaction mechanism and binding mode of phycocyanin to lysozyme: Molecular docking and molecular dynamics simulation.

In this study, multispectral analysis and molecular simulations were performed to investigate the interaction mechanism between phycocyanin (PC) and lysozyme (Lys). The interaction was examined using surface plasmon resonance (SPR), and the structural changes were analyzed using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). The results suggest that the interaction between PC and Lys was primarily driven by electrostatic, hydrophobic, and hydrogen bonding forces. Molecular dynamics (MD) simulation revealed that Lys preferentially binds between the two subunits, alpha (α) and beta (ß), of PC, with residues ASP-13, GLU-106, and GLU-115 on PC and ARG-119, ARG-107, and ARG-98 on Lys being the main contributors to the binding interaction. Additionally, the formation of the PC-Lys complex resulted in increased kinetic and improved thermal stability of PC, which have important implications for PC applications.

Reversible disassembly-reassembly of C-phycocyanin in pressurization-depressurization cycles of high hydrostatic pressure.

Hydrostatic pressure can reversibly modulate protein-protein and protein-chromophore interactions of C-phycocyanin (C-PC) from Spirulina platensis. Small-angle X-ray scattering combined with UV-Vis spectrophotometry and protein modeling was used to explore the color and structural changes of C-PC under high pressure conditions at different pH levels. It was revealed that pressures up to 350 MPa were enough to fully disassemble C-PC from trimers to monomers at pH 7.0, or from monomers to detached subunits at pH 9.0. These disassemblies were accompanied by protein unfolding that caused these high-pressure induced structures to be more extended. These changes were reversible following depressurization. The trimer-to-monomer transition proceeded through a collection of previously unrecognized, L-shaped intermediates resembling C-PC dimers. Additionally, pressurized C-PC showed decayed Q-band absorption and fortified Soret-band absorption. This was evidence that the folded tetrapyrroles, which had folded at ambient pressure, formed semicyclic unfolded conformations at a high pressure. Upon depressurization, the peak intensity and shift all recovered stepwise, showing pressure can precisely manipulate C-PC's structure as well as its color. Overall, a protein-chromophore regulatory theory of C-PC was unveiled. The pressure-tunability could be harnessed to modify and stabilize C-PC's structure and photochemical properties for designing new delivery and optical materials.

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant.

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αß)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 °C), while the pH stability study showed R-PC is stable in the pH range 4-8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on R-PC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads.

Infrared Signatures of Phycobilins within the Phycocyanin 645 Complex.

Aquatic photosynthetic organisms evolved to use a variety of light frequencies to perform photosynthesis. Phycobiliprotein phycocyanin 645 (PC645) is a light-harvesting complex in cryptophyte algae able to transfer the absorbed green solar light to other antennas with over 99% efficiency. The infrared signatures of the phycobilin pigments embedded in PC645 are difficult to access and could provide useful information to understand the mechanism behind the high efficiency of energy transfer in PC645. We use visible-pump IR-probe and two-dimensional electronic vibrational spectroscopy to study the dynamical evolution and assign the fingerprint mid-infrared signatures to each pigment in PC645. Here, we report the pigment-specific vibrational markers that enable us to track the spatial flow of excitation energy between the phycobilin pigment pairs. We speculate that two high-frequency modes (1588 and 1596 cm-1) are involved in the vibronic coupling leading to fast (

Carboxylated chitosan improved the stability of phycocyanin under acidified conditions.

Phycocyanin, a natural blue colorant, derived from Spirulina platensis, is now widely used in the food industry. However, its main drawbacks are loss of color and denature of structure in an acidic environment. In this study, carboxylated chitosan (0.1 %-1 % w/v) was chosen as an additive in acid-denatured phycocyanin for preserving phycocyanin's blue color and natural structure. Zeta-potential and particle size revealed that the carboxylated chitosan with high negative charge adsorbed on phycocyanin and provided stronger electrostatic repulsion to overcome the protein aggregation. Ultraviolet-visible absorption spectrum and fluorescence spectroscopy showed that the carboxylated chitosan recovered the microenvironment of tetrapyrrole chromophores and ß-subunits, which led the secondary structure changed and the trimers depolymerized into the monomers changed by the acidic environment. Furthermore, Fourier transform infrared spectroscopy revealed highly negatively charged carboxylated chitosan with the groups (NH2, COOH and OH) could restored the microenvironment of tetrapyrrole chromophores and ß-subunits of phycocyanin, and interact with phycocyanin through hydrogen bonding, NH bonding, ionic bonding and van der Waals, which led to a change in secondary structure and depolymerization of trimers into monomers. Our study demonstrated the carboxylated chitosan played a beneficial role in recovering the structure of acid-denatured phycocyanin and its blue color.

The Cyanobacterial "Nutraceutical" Phycocyanobilin Inhibits Cysteine Protease Legumain.

The blue biliprotein phycocyanin, produced by photo-autotrophic cyanobacteria including spirulina (Arthrospira) and marketed as a natural food supplement or "nutraceutical," is reported to have anti-inflammatory, antioxidant, immunomodulatory, and anticancer activity. These diverse biological activities have been specifically attributed to the phycocyanin chromophore, phycocyanobilin (PCB). However, the mechanism of action of PCB and the molecular targets responsible for the beneficial properties of PCB are not well understood. We have developed a procedure to rapidly cleave the PCB pigment from phycocyanin by ethanolysis and then characterized it as an electrophilic natural product that interacts covalently with thiol nucleophiles but lacks any appreciable cytotoxicity or antibacterial activity against common pathogens and gut microbes. We then designed alkyne-bearing PCB probes for use in chemical proteomics target deconvolution studies. Target identification and validation revealed the cysteine protease legumain (also known as asparaginyl endopeptidase, AEP) to be a target of PCB. Inhibition of this target may account for PCB's diverse reported biological activities.