Characterization and structural identification of a family 16 carbohydrate-binding module (CBM): First structural insights into porphyran-binding CBM.

Porphyran is a favorable functional polysaccharide widely distributed in Porphyra. It displays a linear structure majorly constituted by alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G) units. Carbohydrate-binding modules (CBMs) are desired tools for the investigation and application of polysaccharides, including in situ visualization, on site and specific assay, and functionalization of biomaterials. However, only one porphyran-binding CBM has been hitherto reported, and its structural knowledge is lacking. Herein, a novel CBM16 family domain from a marine bacterium Aquimarina sp. BL5 was discovered and expressed. The recombinant protein AmCBM16 exhibited the desired specificity for porphyran. Bio-layer interferometry assay revealed that the protein binds to porphyran tetrasaccharide (L6S-G)2 with an association constant of 1.3 × 103 M-1. The structure of AmCBM16 was resolved by the X-ray crystallography, which displays a ß-sandwich fold with two antiparallel ß-sheets constituted by 10 ß-strands. Site-directed mutagenesis analysis demonstrated that the residues Gly-30, Trp-31, Lys-88, Lys-123, Phe-125, and Phe-127 play dominant roles in AmCBM16 binding. This study provides the first structural insights into porphyran-binding CBM.

Red seaweed biorefinery: The influence of sequential extractions on the functional properties of extracted agars and porphyrans.

Red seaweeds are exploited for their hydrocolloids, but other fractions are usually overlooked. In a novel approach, this study aimed to evaluate cold-water (CWE), ethanolic (EE), and alkaline (SE) extractions, alone and in sequence, to simultaneously: i) decrease the hydrocolloid extraction waste (valorizing bioactive side-streams and/or increasing extraction yield); and ii) increase the hydrocolloids' texturizing properties. It is the first time these extractions' synergetic and/or antagonistic effects will be accessed. For Porphyra dioica, a combination of CWE and EE was optimal: a positive influence on the melting temperature (increasing 5 °C to 74 °C) and sulphate content (a 3-fold reduction to 5 %) was observed, compared to a direct porphyran extraction. The same was observed for Gracilaria vermiculophyla, recovering two additional bioactive fractions without impacting the hydrocolloid's extraction (agar with 220 g/cm2 gelling strength and 14 % yield was obtained). The sequential use of CWE, EE, and SE was the most beneficial in Gelidium corneum processing: it enhanced agar's texturizing capacity (reaching 1150 g/cm2, a 1.5-fold increase when compared to a direct extraction), without affecting its 22 % yield or over 88 % purity. Ultimately, these findings clarified the effects of cascading biorefinery approaches from red seaweeds and their pertinence.

Characterization of a GH50 ß-Agarase: A Biotechnological Tool for Preparing Oligosaccharides from Agarose and Porphyran.

Agarase is of vital significance for functional agaro-oligosaccharides production from algal dived agarose. Especially, the exolytic agarases have the advantage of obtaining agaro-oligosaccharides with a specific degree of polymerization. Herein, we cloned and expressed a novel glycoside hydrolase (GH) 50 family ß-agarase OUC-PgJC50 from Photobacterium gaetbulicola. The degradation pattern analysis indicated that OUC-PgJC50 not only showed an exolytic activity with main products of neoagarotetraose from hydrolyzing agarose but also show a hydrolytic activity to transform neoagarotetraose into neoagarobiose. This is the first time that the discovery of a neoagarotetraose-producing exolytic GH50 ß-agarase possesses the activity to transform neoagarotetraose into neoagarobiose, which provided new insight into the recognition of the degradation mode of agarases. Molecular docking and sequence alignment analysis further revealed the His654 residue in OUC-PgJC50 may play a vital role in forming a strong force with l-AHG residue at -4 subsite that helps to produce neoagarotetraose from catalyzing agarose. Moreover, the catalytic ability of OUC-PgJC50 toward another agar polysaccharide porphyran was also described that could hydrolyze porphyran into sulfated oligosaccharides, in which the LA6S-d-Gal was the main products. This study is of vital significance for developing the application range of GH50 ß-agarases.

Pyropia yezoensis porphyran alleviates metabolic disorders via modulating gut microbiota in high-sucrose-fed Drosophila melanogaster.

BACKGROUND: Prebiotics, such as algal polysaccharides, can be used to manage metabolic diseases by modulating gut microbiota. However, the effect of Pyropia yezoensis porphyran (PYP), a red algal polysaccharide, on gut microbiota has not been reported. Thus, the objective of this study was to determine effects of PYP on metabolic disorders caused by high sucrose (HS) and underlying mechanisms involved in such effects. RESULTS: Biochemical analysis demonstrated that an HS diet increased triglyceride and circulating sugar contents (metabolic abnormalities) in Drosophila larvae. It also increased the relative abundance of harmful microbiota within the larvae as identified by 16S ribosomal DNA analysis. PYP supplementation at 25 and 50 g kg-1 equivalently reduced metabolic abnormalities in the HS group. Therefore, 25 g kg-1 PYP was selected to investigate its effects on the metabolic pathway and gut microbiota of larvae in the HS group. The activity of PYP in ameliorating metabolic abnormalities by reverse transcription quantitative real-time polymerase chain reaction analysis was consistent with the expression trend of key factors involved in metabolism regulation. PYP reduced the relative abundance of bacteria causing metabolic abnormalities, such as Escherichia-Shigella and Fusobacterium, but increased the relative abundance of beneficial bacteria such as Bacillus and Akkermansia. However, PYP had no effect on triglyceride and circulating sugar contents in HS-fed larvae treated with a mixture of antibiotics designed to remove gut microbiota. CONCLUSION: PYP exhibits anti-metabolic disorder activity by modulating gut microbiota, thereby supporting the development of PYP as a functional prebiotic derived from red algae food. Copyright © 2022 John Wiley & Sons, Ltd. © 2022 Society of Chemical Industry.

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA-Sepharose CL 4B supports, for the use in bioconversion of residual colorants.

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2 ± 1.3 vs. 12.1 protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure.

BACKGROUND: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. METHODS: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. RESULTS: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein's structural stability. CONCLUSIONS: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

Enzyme-support interactions and inactivation conditions determine Thermomyces lanuginosus lipase inactivation pathways: Functional and florescence studies.

Lipase from Thermomyces lanuginosus (TLL) has been covalently immobilized on heterofunctional octyl-vinyl agarose. That way, the covalently immobilized enzymes will have identical orientation. Then, it has blocked using hexyl amine (HEX), ethylenediamine (EDA), Gly and Asp. The initial activity/stability of the different biocatalysts was very different, being the most stable the biocatalyst blocked with Gly. These biocatalysts had been utilized to analyze if the enzyme activity could decrease differently along thermal inactivation courses depending on the utilized substrate (that is, if the enzyme specificity was altered during its inactivation using 4 different substrates to determine the activity), and if this can be altered by the nature of the blocking agent and the inactivation conditions (we use pH 5, 7 and 9). Results show great changes in the enzyme specificity during inactivation (e.g., activity versus triacetin was much more quickly lost than versus the other substrates), and how this was modulated by the immobilization protocol and inactivation conditions. The difference in the changes induced by immobilization and inactivation were confirmed by fluorescence studies. That is, the functional and structural analysis of partially inactivated immobilized enzyme showed that their inactivation pathway is strongly depended on the support features and inactivation conditions.

Agarose Stearate-Carbomer940 as Stabilizer and Rheology Modifier for Surfactant-Free Cosmetic Formulations.

Some commonly used surfactants in cosmetic products raise concerns due to their skin-irritating effects and environmental contamination. Multifunctional, high-performance polymers are good alternatives to overcome these problems. In this study, agarose stearate (AS) with emulsifying, thickening, and gel properties was synthesized. Surfactant-free cosmetic formulations were successfully prepared from AS and carbomer940 (CBM940) mixed systems. The correlation of rheological parameter with skin feeling was determined to study the usability of the mixed systems in cosmetics. Based on rheological analysis, the surfactant-free cosmetic cream (SFC) stabilized by AS-carbomer940 showed shear-thinning behavior and strongly synergistic action. The SFC exhibited a gel-like behavior and had rheological properties similar to commercial cosmetic creams. Scanning electron microscope images proved that the AS-CBM940 network played an important role in SFC's stability. Oil content could reinforce the elastic characteristics of the AS-CBM940 matrix. The SFCs showed a good appearance and sensation during and after rubbing into skin. The knowledge gained from this study may be useful for designing surfactant-free cosmetic cream with rheological properties that can be tailored for particular commercial cosmetic applications. They may also be useful for producing medicine products with highly viscous or gel-like textures, such as some ointments and wound dressings.

Enhancement of the Antioxidant and Antimicrobial Activities of Porphyran through Chemical Modification with Tyrosine Derivatives.

The chemical modification of porphyran hydrocolloid is attempted, with the objective of enhancing its antioxidant and antimicrobial activities. Sulfated galactan porphyran is obtained from commercial samples of the red algae Porphyra dioica using Soxhlet extraction with water at 100 °C and precipitation with isopropyl alcohol. The extracted porphyran is then treated with modified L-tyrosines in aqueous medium in the presence of NaOH, at ca. 70 °C. The modified tyrosines L1 and L2 are prepared through a Mannich reaction with either thymol or 2,4-di-tert-butylphenol, respectively. While the reaction with 2,4-di-tert-butylphenol yields the expected tyrosine derivative, a mixture of products is obtained with thymol. The resulting polysaccharides are structurally characterized and the respective antioxidant and antimicrobial activities are determined. Porphyran treated with the N-(2-hydroxy-3,5-di-tert-butyl-benzyl)-L-tyrosine derivative, POR-L2, presents a noticeable superior radical scavenging and antioxidant activity compared to native porphyran, POR. Furthermore, it exhibited some antimicrobial activity against S. aureus. The surface morphology of films prepared by casting with native and modified porphyrans is studied by SEM/EDS. Both POR and POR-L2 present potential applicability in the production of films and washable coatings for food packaging with improved protecting characteristics.

Porphyran-derived oligosaccharides alleviate NAFLD and related cecal microbiota dysbiosis in mice.

Porphyran and its derivatives possess a variety of biological activities, such as ameliorations of oxidative stress, inflammation, hyperlipemia, and immune deficiencies. In this study, we evaluated the potential efficacy of porphyran-derived oligosaccharides from Porphyra yezoensis (PYOs) in alleviating nonalcoholic fatty liver disease (NAFLD) and preliminarily clarified the underlying mechanism. NAFLD was induced by a high-fat diet for six months in C57BL/6J mice, followed by treatment with PYOs (100 or 300 mg/kg/d) for another six weeks. We found that PYOs reduced hepatic oxidative stress in mice with NAFLD, which plays a critical role in the occurrence and development of NAFLD. In addition, PYOs could markedly decrease lipid accumulation in liver by activating the IRS-1/AKT/GSK-3ß signaling pathway and the AMPK signaling pathway in mice with NAFLD. PYOs also apparently relieved the hepatic fibrosis induced by oxidative stress via downregulation of TGF-ß and its related proteins, so that liver injury was markedly alleviated. Furthermore, PYOs treatment relieved cecal microbiota dysbiosis (such as increasing the relative abundance of Akkermansia, while decreasing the Helicobacter abundance), which could alleviate oxidative stress, inflammation, and lipid metabolism, and protect the liver to a certain degree. In summary, PYOs treatment remarkably improved NAFLD via a specific molecular mechanism and reshaped the cecal microbiota.

ε­Polylysine-stabilized agarose/polydopamine hydrogel dressings with robust photothermal property for wound healing.

Polydopamine (PDA) is emerging as an attractive photothermal agent due to its good photothermal performance and excellent biocompatibility. However, without chemical modification, PDA is normally unstable and usually leached out from the constructed biomaterials, realistically limiting its application space. Here, we constructed a new hydrogel dressing with robust and stable photothermal performance by introduction of ε-Polylysine (ε-PL) into agarose/PDA matrix to efficiently lock PDA. By optimizing PDA/ε-PL rational dose in agarose network structure, a hybrid agarose/PDA/ε-PL hydrogel (ADPH) with stable photothermal functionality and desirable physicochemical properties could be achieved. ADPH possessed satisfactory microbicidal efficacy in vivo, which enabled the bacteria-infected skin wound to be cured quickly by successful suppressing inflammation, accelerating collagen deposition and promoting angiogenesis in a bacterial-infected wound model. Collectively, this study illustrates a simple, convenient but powerful strategy to design functionally stable ADPH dressing for treating dermal wounds, which could open vistas in clinical wound management.

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies.

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile.

Ameliorating quercetin constraints in cancer therapy with pH-responsive agarose-polyvinylpyrrolidone -hydroxyapatite nanocomposite encapsulated in double nanoemulsion.

Despite quercetin (QC) promising features for cancer therapy, low solubility, poor permeability, and short biological half-life time significantly confine its application in cancer therapy. In this study, a novel approach is developed to improve loading efficiency and attain quercetin sustained-release concurrently. In this direction, hydrogel nanocomposite of agarose (AG)-polyvinylpyrrolidone (PVP)-hydroxyapatite (HAp) was loaded with QC. Incorporating HAp nanoparticles in the AG-PVP hydrogel improved the loading efficiency up to 61%. Also, the interactions between nanoparticle, drug, and hydrogel polymers rendered the nanocomposite pH-responsive at acidic conditions and controlled the burst release at neutral conditions. Then, QC-loaded hydrogel was encapsulated into the water in oil in water nanoemulsions to further sustain the drug release. As a result, the pH-responsive release of QC with prolonged-release over 96 h was observed. In more detail, according to the Korsmeyer-Peppas mathematical model, the mechanism of release was anomalous (diffusion-controlled) at pH 7.4 and anomalous transport (dissolution-controlled) at pH 5.4. The presence of all nanocomposite components was confirmed with FTIR analysis, and XRD results approved the incorporation of QC in the fabricated nanocomposite. The homogeneous surface of the nanocomposite in FESEM images showed good compatibility between components. The zeta potential analysis confirmed the good stability of the nanocarriers. Besides, the fabricated AG-PVP-HAp-QC platform showed significant cytotoxicity on MCF-7 cells compared to QC as a free drug (p < 0.001) and to quercetin-loaded AG-PVP (AG-PVP-QC) (p < 0.001) with enhanced apoptosis induction after the addition of HAp. Accordingly, this delivery platform ameliorated loading and sustained-release of QC, as well as its anticancer activity by releasing the drug at an effective therapeutic level over a long period to induce apoptosis. Thus, turning this drug delivery system into a potential candidate for further biomedical applications.

How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the Sec61 channel subunit Sbh1 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent signal peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.

Antidepressant-like Effects of Degraded Porphyran Isolated from Porphyra haitanensis.

INTRODUCTION: Degraded porphyran is a bioactive polysaccharide extracted from Porphyra haitanensis (P. haitanensis). According to the previous studies, it produced anti-inflammatory activity, but little is known about its effects on depression. METHODS AND RESULTS: As inflammation is one of the critical factors involved in the development of depression, this study aims to elucidate the potential antidepressant-like effects of degraded porphyran. The results show that acute porphyran treatment decreased the immobility time in despair tests. In addition, subchronic porphyran administration reverses depressive-like behaviors in lipopolysaccharide (LPS)-treated mice. Meanwhile, porphyran inhibits NF-κB/NLRP3 signaling, proinflammatory cytokine release, and microglial activation in the hippocampus. Moreover, chronic porphyran treatment activates hippocampal brain derived neurotrophic factor (BDNF)/TrkB/ERK/CREB signaling pathway in chronic unpredictable mild stress (CUMS) in mice. As a result, neurogenesis and spinogenesis are maintained. CONCLUSIONS: The findings of the present study indicate that degraded porphyran intake provides a potential strategy for depression treatment, which is mediated by the inhibition of neuroinflammation and the enhancement of neurogenesis and spinogenesis in the central nervous systems.

Porphyran and oligo-porphyran originating from red algae Porphyra: Preparation, biological activities, and potential applications.

Porphyra is one of the most economically important red algae in the world. The functional components extracted from Porphyra such as porphyrans, proteins, lipids, and minerals have strong physiological activities. Porphyran, a sulfated galactan, is composed of alternating 1,4-linked α-l-galactopyranose-6-sulfate (L6S) and 1,3-linked ß-d-galactopyranose (G). Porphyran and oligo-porphyran have a series of pharmacological and biological functions, such as antioxidation, anticancer, antiaging, antiallergic, immunomodulatory, hypoglycaemic, and hypolipidemic effects. Thus, red algae Porphyra-derived porphyran and oligo-porphyran have various potential applications in food, medicine, and cosmetic fields. For better application, this review introduces and summarizes the structure and source of porphyran as well as the preparation methods, biological activities, and potential applications of porphyran and oligo-porphyran. Moreover, the future research directions and emphasis of porphyran and oligo-porphyran preparation as well as their functional activities and applications are highlighted and prospected.

Optimization of Porphyran Extraction from Pyropia yezoensis by Response Surface Methodology and Its Lipid-Lowering Effects.

Macroalgae polysaccharides are phytochemicals that are beneficial to human health. In this study, response surface methodology was applied to optimize the extraction procedure of Pyropia yezoensis porphyran (PYP). The optimum extraction parameters were: 100 °C (temperature), 120 min (time), and 29.32 mL/g (liquid-solid ratio), and the maximum yield of PYP was 22.15 ± 0.55%. The physicochemical characteristics of PPYP, purified from PYP, were analyzed, along with its lipid-lowering effect, using HepG2 cells and Drosophila melanogaster larvae. PPYP was a ß-type sulfated hetero-rhamno-galactan-pyranose with a molecular weight of 151.6 kDa and a rhamnose-to-galactose molar ratio of 1:5.3. The results demonstrated that PPYP significantly reduced the triglyceride content in palmitic acid (PA)-induced HepG2 cells and high-sucrose-fed D. melanogaster larvae by regulating the expression of lipid metabolism-related genes, reducing lipogenesis and increasing fatty acid ß-oxidation. To summarize, PPYP can lower lipid levels in HepG2 cells and larval fat body (the functional homolog tissue of the human liver), suggesting that PPYP may be administered as a potential marine lipid-lowering drug.

Synthesis and Evaluation of Dye-Ligand Affinity Adsorbents for Protein Purification.

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity toward a large number of proteins. They are commercially available, inexpensive, stable and can easily be immobilized. Significant factors that contribute to the successful operation of a dye-ligand chromatography include matrix type, dye-ligand density, adsorption along with elution conditions and flow rate. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a given dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris on Cibacron Blue 3GA-Sepharose affinity adsorbent is given as an example.

Heparin-Binding Affinity Tag: A Novel Affinity Tag for Simple and Efficient Purification of Recombinant Proteins.

Heparin, a polysulfated polyanionic member of the glycosaminoglycan family, is known to specifically bind to a number of functionally important proteins. Based on the available information on structural specificity of heparin-protein interactions, a novel heparin-binding peptide (HB) affinity tag has been designed to achieve simple and cost-effective purification of target recombinant proteins. The HB-fused recombinant target proteins are purified on a heparin-Sepharose column using a stepwise/continuous sodium chloride gradient. A major advantage of the HB tag is that the HB-fused target proteins can be purified under denaturing conditions in the presence of 8 M urea. In addition, polyclonal antibody directed against the HB tag can be used to specifically detect and quantitate the HB-fused recombinant protein(s). Herein, a step-by-step protocol(s) for the purification of different soluble recombinant target proteins is described. In addition, useful tips to troubleshoot potential problems and also suggestions to successfully adopt the HB-tag-based purification to a wide range of target proteins are provided.

Porphyran-capped silver nanoparticles as a promising antibacterial agent and electrode modifier for 5-fluorouracil electroanalysis.

In the present work, the green synthesis of silver nanoparticles (AgNPs) using the sulfated polysaccharide porphyran (PFR) as capping agent and d-glucose as reducing agent is described. PFR was extracted from red seaweed and characterized by employing 13C NMR and determination of total sugar, protein, and sulfate contents. The obtained AgNPs-PFR were characterized by using UV-VIS spectroscopy, zeta potential determination, FESEM, and TEM, which demonstrated that PFR was effective at capping the AgNPs, yielding stable suspensions. The AgNPs-PFR presented good antimicrobial properties against Gram-positive and Gram-negative bacterial strains (Staphylococcus aureus and Escherichia coli, respectively). The AgNPs-PFR were also employed as the modifier of carbon paste electrodes, which were efficiently applied as electrochemical sensors for the determination of 5-fluorouracil (5-FU), an important anticancer drug, through square wave voltammetry (SWV). The AgNPs-PFR improved the electrochemical properties of the electrodes, and enhanced their electroanalytical performance. The developed sensing device presented detection and quantification limits equal to 10.7 and 35.8 µmol L-1, respectively, towards 5-FU determination. The proposed electrochemical sensor successfully quantified 5-FU in a real pharmaceutical formulation, confirming its potential as a new promising analytical detection tool for 5-FU quality control purposes.