In vitro digestibility and Caco-2 cell bioavailability of sea lettuce (Ulva fenestrata) proteins extracted using pH-shift processing.

Seaweed is a promising sustainable source of vegan protein as its farming does not require arable land, pesticides/insecticides, nor freshwater supply. However, to be explored as a novel protein source the content and nutritional quality of protein in seaweed need to be improved. We assessed the influence of pH-shift processing on protein degree of hydrolysis (%DH), protein/peptide size distribution, accessibility, and cell bioavailability of Ulva fenestrata proteins after in vitro gastrointestinal digestion. pH-shift processing of Ulva, which concentrated its proteins 3.5-times, significantly improved the %DH from 27.7±2.6% to 35.7±2.1% and the amino acid accessibility from 56.9±4.1% to 72.7±0.6%. Due to the higher amino acid accessibility, the amount of most amino acids transported across the cell monolayers was higher in the protein extracts. Regarding bioavailability, both Ulva and protein extracts were as bioavailable as casein. The protein/peptide molecular size distribution after digestion did not disclose a clear association with bioavailability.

Cyanobacteria and Eukaryotic Microalgae as Emerging Sources of Antibacterial Peptides.

Cyanobacteria and microalgae are oxygen-producing photosynthetic unicellular organisms encompassing a great diversity of species, which are able to grow under all types of extreme environments and exposed to a wide variety of predators and microbial pathogens. The antibacterial compounds described for these organisms include alkaloids, fatty acids, indoles, macrolides, peptides, phenols, pigments and terpenes, among others. This review presents an overview of antibacterial peptides isolated from cyanobacteria and microalgae, as well as their synergism and mechanisms of action described so far. Antibacterial cyanopeptides belong to different orders, but mainly from Oscillatoriales and Nostocales. Cyanopeptides have different structures but are mainly cyclic peptides. This vast peptide repertoire includes ribosomal and abundant non-ribosomal peptides, evaluated by standard conventional methodologies against pathogenic Gram-negative and Gram-positive bacteria. The antibacterial activity described for microalgal peptides is considerably scarcer, and limited to protein hydrolysates from two Chlorella species, and few peptides from Tetraselmis suecica. Despite the promising applications of antibacterial peptides and the importance of searching for new natural sources of antibiotics, limitations still persist for their pharmaceutical applications.

Photooxidative stress-inducible orange and pink water-soluble astaxanthin-binding proteins in eukaryotic microalga.

Lipid astaxanthin, a potent antioxidant known as a natural sunscreen, accumulates in eukaryotic microalgae and confers photoprotection. We previously identified a photooxidative stress-inducible water-soluble astaxanthin-binding carotenoprotein (AstaP) in a eukaryotic microalga (Coelastrella astaxanthina Ki-4) isolated from an extreme environment. The distribution in eukaryotic microalgae remains unknown. Here we identified three novel AstaP orthologs in a eukaryotic microalga, Scenedesmus sp. Oki-4N. The purified proteins, named AstaP-orange2, AstaP-pink1, and AstaP-pink2, were identified as secreted fasciclin proteins with potent 1O2 quenching activity in aqueous solution, which are characteristics shared with Ki-4 AstaP. Nonetheless, the absence of glycosylation in the AstaP-pinks, the presence of a glycosylphosphatidylinositol (GPI) anchor motif in AstaP-orange2, and highly acidic isoelectric points (pI = 3.6-4.7), differed significantly from that of AstaP-orange1 (pI = 10.5). These results provide unique examples on the use of water-soluble forms of astaxanthin in photosynthetic organisms as novel strategies for protecting single cells against severe photooxidative stresses.

Innovative processing strategies and technologies to obtain hydrocolloids from macroalgae for food applications.

The macroalgal hydrocolloid industry is a rapidly growing industry with an annual growth in the global production of 2-3 %. Hydrocolloids from macroalgae are mainly in the form of polysaccharides with other nutrients, such as vitamins, minerals, and proteins. Due to its potential industrial applications, macroalgae have been used as raw materials for hydrocolloids extraction. Compared to the conventional extraction methods, emerging innovative assisted extraction technologies (e.g., Ultrasound/Microwave assisted extraction) have been developed to maximise the extraction yields, efficiency and thereby, maintaining sustainability along the process. These novel techniques are considered as clean and green strategies, with a potential for a large-scale production; thus, avoiding or reducing the use of chemicals. However, more research is required to establish their mechanism of action in order to finally implement them at industrial level. This paper reviews the most relevant strategies and technologies involved in the production of hydrocolloids from macroalgae.

Methods for Algal Protein Isolation and Proteome Analysis.

Microalgae present promising feedstocks to produce renewable fuel and chemical intermediates, in part due to high storage carbon flux capacity to triacylglycerides or storage carbohydrates upon nutrient deprivation. However, the mechanism(s) governing deprivation-mediated carbon partitioning remain to be fully elucidated, limiting targeted strain engineering strategies in algal biocatalysts. Though genomic and transcriptomic analyses offer key insights into these mechanisms, active post-transcriptional regulatory mechanisms, ubiquitous in many microalgae, necessitate proteomic and post-translational (e.g., phospho- and nitroso-proteomic) analyses to more completely evaluate algal responsiveness to nutrient deprivation. Herein, we describe methods for isolating total algal protein and conducting proteomic, phosphoproteomic, and nitrosoproteomic analyses. We focus on methods deployed for the chlorophyte, Chlorella vulgaris, a model oleaginous alga with high flux to renewable fuel and chemical precursors.

Localization, Extraction, and Quantification of Plant and Algal Arabinogalactan Proteins.

Arabinogalactan proteins are a diverse group of cell wall-associated proteoglycans. While structural and molecular genetic analyses have contributed to the emerging improved understanding of the wide-range of biological processes in which AGPs are implicated; the ability to detect, localize, and quantify them is fundamentally important. This chapter describes three methods: histological staining, radial gel diffusion, and colorimetric quantification, each of which utilize the ability of Yariv reagent to bind to AGPs.

In Silico Analysis of ACE Inhibitory Peptides from Chloroplast Proteins of Red Alga Grateloupia asiatica.

Inhibition of angiotensin I-converting enzyme (ACE) is one of the key factors to repress high blood pressure. Although many studies have been reported that seaweed protein hydrolysates showed the ACE inhibitory activity, the comprehensive understanding of the relationship was still unclear. In this study, we employed chloroplast genome for in silico analysis and compared it with in vitro experiments. We first extracted water-soluble proteins (WSP) from red alga Grateloupia asiatica, which contained mainly PE, PC, APC, and Rbc, and prepared WSP hydrolysate by thermolysin, resulting that the hydrolysate showed ACE inhibitory activity. Then, we determined the complete chloroplast genome of G. asiatica (187,518 bp: 206 protein-coding genes, 29 tRNA, and 3 rRNA) and clarified the amino acid sequences of main WSP, i.e., phycobiliproteins and Rubisco, to perform in silico analysis. Consequently, 190 potential ACE inhibitory peptides existed in the main WSP sequences, and 21 peptides were obtained by in silico thermolysin digestion. By comparing in vitro and in silico analyses, in vitro ACE inhibitory activity was correlated to the IC50 value from in silico digestion. Therefore, in silico approach provides insight into the comprehensive understanding of the potential bioactive peptides from seaweed proteins.

Phycocyanin Extracted from Oscillatoria minima Shows Antimicrobial, Algicidal, and Antiradical Activities: In silico and In vitro Analysis.

BACKGROUND: Phycocyanin is an algae-derived protein, which binds to pigment for harvesting light. It has been reported in various different species, including that of red algae, dinoflagellates, and cryptophyta. Importantly, phycocyanin has enormous applications, including cosmetic colorant, food additive, biotechnology, diagnostics, fluorescence detection probe, an anticancer agent, anti-inflammatory, immune enhancer, etc. In addition, several different algae were utilized for the isolation of cyano-phycocyanin (C-PC), but most of the purification methods consist of several steps of crude extraction. AIM: To isolate C-PC from a new source of microalgae with better purity level and to evaluate its antimicrobial, algicidal, and antiradical activities. METHODS: Biological activity, permeability, pharmacokinetics, and toxicity profile of C-PC were predicted by in silico studies. C-PC was purified and isolated by using ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration chromatography. C-PC was characterized by SDS-PAGE and elution profile (purity ratio) analysis. Antimicrobial and algicial activities of C-PC were evaluated by the microtitre plate based assays. Antiradical activity of C-PC was evaluated by DPPH- and ABTS*+ radical scavenging assays. CONCLUSION: C-PC was extracted from Oscillatoria minima for the first time, followed by its quantitative as well qualitative evaluation, indicating a new alternative source of this important protein. Furthermore, the antimicrobial, algicidal, and antiradical activities of the isolated C-PC extract have been demonstrated by both in silico as well as in vitro methods.

Extraction, structural and functional properties of Haematococcus pluvialis protein after pigment removal.

Protein from Haematococcus pluvialis (H. pluvialis) residues after pigment removal was prepared by alkaline extraction and acid precipitation. And the structural and functional properties of protein extracts were measured and analyzed. The effect of extraction conditions (liquid/solid ratio 10-20 mL/g), pH 10-12 and 25-45 °C (temperature) on the yield of H. pluvialis protein (HP) was carried out using Box-Behnken design. Under optimum extraction conditions: liquid/solid 20 mL/g, pH 11.5 and lower extraction temperature 35 °C, the highest extraction yield (81.36%) of HP was gained, which was close to the predicted value (83.32%). The HP exhibited the better functional properties. The solubility could reach 93.65% at pH 10.0; the foaming capacity and stability were 88.32 and 89.62%, respectively; the emulsifying capacity and stability were 161.52 and 48.2%, respectively; the water holding capacity (WHC) and oil absorption capacity (OAC) were 4.06 and 3.29 g/g, respectively. Fourier Transform infrared spectroscopy (FTIR) data of the HP showed the ß-sheet content (30.37%) was the maximum, while α-helix content (14.86%) was the minimum, whereas the content of amino acids was rich in HP. These results demonstrated that the HP had potential use in food industry.

Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway.

Spirulina is a type of filamentous blue-green microalgae known to be rich in nutrients and to have pharmacological effects, but the effect of spirulina on the small intestine epithelium is not well understood. Therefore, this study aims to investigate the proliferative effects of spirulina crude protein (SPCP) on a rat intestinal epithelial cells IEC-6 to elucidate the mechanisms underlying its effect. First, the results of wound-healing and cell viability assays demonstrated that SPCP promoted migration and proliferation in a dose-dependent manner. Subsequently, when the mechanisms of migration and proliferation promotion by SPCP were confirmed, we found that the epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAPK) signaling pathways were activated by phosphorylation. Cell cycle progression from G0/G1 to S phase was also promoted by SPCP through upregulation of the expression levels of cyclins and cyclin-dependent kinases (Cdks), which regulate cell cycle progression to the S phase. Meanwhile, the expression of cyclin-dependent kinase inhibitors (CKIs), such as p21 and p27, decreased with SPCP. In conclusion, our results indicate that activation of EGFR and its downstream signaling pathway by SPCP treatment regulates cell cycle progression. Therefore, these results contribute to the research on the molecular mechanism for SPCP promoting the migration and proliferation of rat intestinal epithelial cells.

Isolation and characterization of a tandem-repeated cysteine protease from the symbiotic dinoflagellate Symbiodinium sp. KB8.

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH4)2SO4 fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography-coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31-32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Integration Process for Protein Extraction from Microalgae Using Liquid Biphasic Electric Flotation (LBEF) System.

Microalgae are the most promising sources of protein, which have high potential due to their high-value protein content. Conventional methods of protein harnessing required multiple steps, and they are generally complex, time consuming, and expensive. Currently, the study of integration methods for microalgae cell disruption and protein recovery process as a single-step process is attracting considerable interest. This study aims to investigate the novel approach of integration method of electrolysis and liquid biphasic flotation for protein extraction from wet biomass of Chlorella sorokiniana CY-1 and obtaining the optimal operating conditions for the protein extraction. The optimized conditions were found at 60% (v/v) of 1-propanol as top phase, 250 g/L of dipotassium hydrogen phosphate as bottom phase, crude microalgae loading of 0.1 g, air flowrate of 150 cc/min, flotation time of 10 min, voltage of 20 V and electrode's tip touching the top phase of LBEF. The protein recovery and separation efficiency after optimization were 23.4106 ± 1.2514% and 173.0870 ± 4.4752%, respectively. Comparison for LBEF with and without the aid of electric supply was also conducted, and it was found that with the aid of electrolysis, the protein recovery and separation efficiency increased compared to the LBEF without electrolysis. This novel approach minimizes the steps for overall protein recovery from microalgae, time consumption, and cost of operation, which is beneficial in bioprocessing industry.

Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

Functional Expression and Characterization of the Recombinant N-Acetyl-Glucosamine/N-Acetyl-Galactosamine-Specific Marine Algal Lectin BPL3.

Lectins, characterized by their carbohydrate-binding ability, have extensive practical applications. However, their industrial use is limited due to impurity. Thus, quality-controlled production of recombinant lectin is necessary. In this study, the algal lectin BPL3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocyte. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled large-scale production of recombinant lectins.

Multi-Product Microalgae Biorefineries: From Concept Towards Reality.

Although microalgae are a promising biobased feedstock, industrial scale production is still far off. To enhance the economic viability of large-scale microalgae processes, all biomass components need to be valorized, requiring a multi-product biorefinery. However, this concept is still too expensive. Typically, downstream processing of industrial biotechnological bulk products accounts for 20-40% of the total production costs, while for a microalgae multi-product biorefinery the costs are substantially higher (50-60%). These costs are high due to the lack of appropriate and mild technologies to access the different product fractions such as proteins, carbohydrates, and lipids. To reduce the costs, simplified processes need to be developed for the main unit operations including harvesting, cell disruption, extraction, and possibly fractionation.

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H2) production and H2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity).

Photoprotection strategies of the alga Nannochloropsis gaditana.

Nannochloropsis spp. are algae with high potential for biotechnological applications due to their capacity to accumulate lipids. However, little is known about their photosynthetic apparatus and acclimation/photoprotective strategies. In this work, we studied the mechanisms of non-photochemical quenching (NPQ), the fast response to high light stress, in Nannochloropsis gaditana by "locking" the cells in six different states during quenching activation and relaxation. Combining biochemical analysis with time-resolved fluorescence spectroscopy, we correlated each NPQ state with the presence of two well-known NPQ components: de-epoxidized xanthophylls and stress-related antenna proteins (LHCXs). We demonstrated that after exposure to strong light, the rapid quenching that takes place in the antennas of both photosystems was associated with the presence of LHCXs. At later stages, quenching occurs mainly in the antennas of PSII and correlates with the amount of de-epoxidised xanthophylls. We also observed changes in the distribution of excitation energy between photosystems, which suggests redistribution of excitation between photosystems as part of the photo-protective strategy. A multistep model for NPQ induction and relaxation in N. gaditana is discussed.

Isolation and identification of anti-proliferative peptides from Spirulina platensis using three-step hydrolysis.

BACKGROUND: Spirulina platensis is an excellent source of proteins (>60%) that can be hydrolyzed into bioactive peptides. RESULTS: In this study, whole proteins of Spirulina platensis were extracted and hydrolyzed using three gastrointestinal endopeptidases (pepsin, trypsin and chymotrypsin). Subsequently, gel filtration chromatography was employed to separate hydrolysates, and four fractions (Tr1-Tr4) were obtained. Among them, Tr2 showed the strongest anti-proliferation activities on three cancer cells (MCF-7, HepG-2 and SGC-7901), with IC50 values of <31.25, 36.42 and 48.25 µg mL-1 , respectively. Furthermore, a new peptide, HVLSRAPR, was identified from fraction Tr1. This peptide exhibited strong inhibition on HT-29 cancer cells with an IC50 value of 99.88 µg mL-1 . CONCLUSION: Taken together, these peptides possessed anti-proliferation activities on cancer cells and low cytotoxicity on normal cells, suggesting that they might serve as a natural anticancer agent for nutraceutical and pharmaceutical industries. © 2016 Society of Chemical Industry.

Late Somatic Gene 2 disrupts parental spheroids cooperatively with Volvox hatching enzyme A in Volvox.

MAIN CONCLUSION: We identified LSG2 as a novel lytic enzyme that accumulates in the parental extracellular matrix and disrupts parental spheroids cooperatively with VheA secreted by juveniles in Volvox. Spatiotemporally restricted degradation of extracellular matrix (ECM) is essential for development and survival in multicellular organisms. In an asexual life cycle of green algae Volvox, juveniles are released from parental spheroids through holes made by restricted degradation of parental ECM at the proper timing. Lytic enzyme(s) should specifically degrade parental ECM upon Volvox hatching, but little is known about the mechanisms of spatiotemporally restricted parental degradation. Here, we identified a glycoprotein encoded by the Late Somatic Gene 2 (LSG2) as a novel lytic enzyme that accumulates in parental ECM during the prehatching stages. The dual action of LSG2 and Volvox hatching enzyme A (VheA), a serine protease secreted by juveniles, causes the degradation of ECM sheets at all stages and destroys even daughter spheroids, while VheA alone disrupts spheroids only in the prehatching stage when LSG2 is accumulated, suggesting that the combination of LSG2 and VheA is sufficient to cause the degradation of ECM sheet. In the prehatching stage, parental spheroids became susceptible to the proteolysis by a mixture of bacterial proteases applied externally, which could be facilitated by LSG2. These results suggest that LSG2 disrupts parental ECM cooperatively with VheA by modifying the parental ECM to make it fragile, and that the appropriate activity of these enzymes is crucial for the parent-specific ECM degradation at the proper timing.