Mannuronic Acid Research Articles

Pattern of Specific Oxidation of Konjac Glucomannan with TEMPO/NaBr/NaClO system

The TEMPO/NaBr/NaClO system was used to modify konjac glucomannan and prepare β-1,4-linked D-mannuronic/glucuronic acid oligosaccharides. The impact of oxidant amount on the degree of oxidation, molecular weight, terminal structure and the ratio of sugar units were systematically investigated. Mannoses were transformed to mannuronic acids quantitatively, while some glucose were transformed to glucuronic acids, some degraded. The unspecific oxidation causes strong degradation of konjac glucomannan sugar chains. Multiple terminal structures were observed in the oxidized KGM products. According to those observations, the oxidation pattern of konjac glucomannan with the TEMPO/NaBr/NaClO system was speculated. It is suggested that the axial hydroxyl group at position 2 in mannose might form a hydrogen bond with the oxygen on the sugar ring. It would against the unspecific oxidation. While, both specific and unspecific oxidations were observed at glucose units with an equatorial hydroxyl group at position 2, and the degradation occurs mostly on glucose during the oxidation.

A novel alginate from Sargassum seaweed promotes diabetic wound healing by regulating oxidative stress and angiogenesis

Diabetic skin ulcer is one of the most severe complications in diabetes, however, current therapeutic approaches are not effective enough. Agents modulating oxidative stress, inflammation, and angiogenesis are quite promising for alleviation of diabetic skin ulcers. In this study, a novel Sargassum kjellmanianum-derived polysaccharide (SARP) was prepared. SARP was an alginate with Mw of 45.4 kDa, consisting of 76.56% mannuronic acid, 18.89% guluronic acid, and 4.55% glucuronic acid. SARP could attenuate oxidative stress-induced cell damage via activating nuclear factor erythroid 2-related factor 2 (Nrf2). SARP also promoted the migration and tube formation of HUVECs, which was related to the increased vascular endothelial growth factor (VEGF) expression. In diabetic wound model, SARP (iv, 200 mg/kg) administration increased angiogenesis, alleviated oxidative stress, ameliorated diabetes-related aberrations, and thereby accelerated diabetic wound healing. These findings identified SARP had potential to be developed as a drug candidate for diabetic skin ulcers.

Sulfated heteropolysaccharides from Undaria pinnatifida: Structural characterization and transcript-metabolite profiling of immunostimulatory effects on RAW264.7 cells.

Investigation of the polysaccharides from an edible marine brown algae Undaria pinnatifida has led to obtaining three fractional sulfated polysaccharides UPPs 1-3 with molecular weights of 7.212kDa, 13.924kDa, and 55.875kDa, respectively. All UPPs are composed of galactose, xylose, glucose, mannose, glucuronic acid, and mannuronic acid, while UPP-3 also consisted of fucose, arabinose, and fructose. The immunostimulatory assay revealed that UPP-3 had important effects on cell viability, nitric oxide levels, and secretion of cytokines TNF-α and IL-6 in RAW264.7 cells. Furthermore, the transcript-metabolite data along with western blot and immunofluorescent staining revealed that UPP-3 could stimulate the Toll-like receptor (TLR4) associated with the nuclear factor kappa-B (NF-κB) p65 signaling pathway of RAW264.7 cells. These findings of the immunomodulatory sulfated polysaccharide will provide a basic understanding to further exploitation of U. pinnatifida polysaccharides.

Evaluating Mannuronic Acid Effect on Gene Expression Profile of Inflammatory Mediators in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is a multisystem disorder. Various studies have shown the important role of inflammatory factors tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-22, MYD88, and toll-like receptor 2 (TLR2) in this disease. In this study, we investigated the anti-inflammatory effects of B-D-Mannuronic acid (M2000), as a new immunosuppressive drug, on the expression of these inflammatory markers in peripheral blood mononuclear cells (PBMCs) of RA patients. The blood samples of active RA patients and healthy volunteers were used for PBMCsl separation. The cells were cultured with LPS (1 µg/mL), low (5 µg/mL), moderate (25 µg/mL), and high (50 µg/mL) doses of M2000 and a single dose of diclofenac (1 µg/mL) to evaluate TNF-α, IL-6, IL-22, MYD88, and TLR2 genes expression by quantitative real-time (qRT-PCR). Cell surface expression and MFI of TLR2 were assessed; using flow cytometry. Our findings exhibited a significant reduction of TNF-α, IL-6, and MYD88 gene expressions after treatment with three doses of M2000 and an optimum dose of diclofenac. TLR2 gene expression was significantly diminished by moderate and high doses of M2000 and a single dose of diclofenac. Moreoversurface expression of TLR2 was significantly downregulated by moderate and high doses of M2000, while MFI of this receptor was significantly reduced by three doses of M2000. The results of this research showed that M2000 was able to significantly reduce the gene expression of inflammatory molecules TNF-α, IL-6, MYD88, and TLR2 in patients PBMCs. factor-alpha; Rheumatoid arthritis. These data revealed a part of the molecular mechanisms of M2000 in the treatment process.

The Effects of Mannuronic Acid on IL-1β, IL-17A, STAT1, and STAT3 Gene Expressions and TLR2 and TLR4 Molecules in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic neurologic disease defined by inflammation and demyelination of the central nervous system that comes with variable degrees of axonal and neuronal damage. The efficacy of β-D-mannuronic acid (M2000) as a novel drug with immunosuppressive properties (patented: PCT/EP2017/067920), has been shown in an experimental model of MS. In this study, the effects of M2000 on interleukin (IL)-1β, IL-17A, signal transducer and activator of transcription (STAT) 1, and STAT3 gene expressions and Toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) molecules in patients with secondary progressive MS were evaluated. In this study, 14 patients with secondary progressive MS and 14 healthy subjects (as control group) were entered from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). The gene expressions of IL-1β, IL-17A, STAT1, and STAT3 were assessed at the baseline and then measured after 6months of therapy with M2000 by using the quantitative real-time polymerase chain reaction method. Moreover, the expressions of TLR2 and TLR4 molecules on peripheral blood mononuclear cells were evaluated by the flow cytometry method. The gene expressions of IL-17A, STAT1, and STAT3 in patients with MS decreased after 6months of therapy with M2000 comparing before treatment. Also, the gene expression of IL-1β decreased numerically after 6months. Furthermore, the expressions of TLR2 and TLR4 on PBMCs of the patients declined when compared to baseline. The results of this investigation revealed that M2000 could downregulate IL-17, STAT1, and STAT3 genes in patients with secondary progressive MS and also reduce the expressions of TLR2 and TLR4 on PBMCs. Moreover, M2000 declined numerically IL-β gene expression.

Tau and amyloid beta differentially affect the innate immune genes expression in Drosophila models of Alzheimer's disease and β- D Mannuronic acid (M2000) modulates the dysregulation.

Alzheimer's disease (AD) is the most common cause of dementia and neuroinflammation is considered as one of the main culprits. The aim of this study was to evaluate the independent role of Aβ42 and tau on the inflammatory pathway in the Drosophila models of AD and investigating the potential modulating effect of M2000 as a novel NSAIDs in those flies. The expression levels of relish, orthologs of NF-κB, antimicrobial peptide (AMP) including attacin A, diptericin B and a dual oxidase (Duox) as a ROS mediator, were evaluated in both M2000 treated and untreated groups followed by brain histology analysis to assess the extent of neurodegeneration. The potential inhibitory role of M2000 (β-D Mannuronic acid) on the aggregation of tau protein was also investigated in vitro. According to the result, there was a significant induction of Duox, AMPs and its transcription factor expression in both aged and Drosophila models of AD which was in accordance with the increase in the number of vacuoles in the brain section of Drosophila models of AD. Interestingly M2000 treatment revealed a significant reduction in all neurodegeneration indexes; in vivo and anti-aggregating property; in vitro. Findings suggest that M2000 has potential to be an AD therapeutic agent.

Simultaneous recovery of phosphorus and alginate-like exopolysaccharides from two types of aerobic granular sludge

Wastewater treatment plants are expected to realize not only pollutants removal from wastewater but also resources recovery such as phosphorus (P) and alginate-like exopolysaccharides (ALE) from the produced sludge. In this study, ALE extraction and fractionation from the same activated sludge-derived bacterial aerobic granular sludge (AGS) and algal-bacterial AGS were performed in addition to P fate examination during ALE recovery. Results showed that the ALE content recovered from algal-bacterial AGS was 8.81 ± 0.02 mg/g-volatile suspended solids (VSS), about 2.8 times higher than that from bacterial AGS when fed with the same synthetic wastewater. Moreover, the mannuronic acid to guluronic acid (MG) blocks accounted for the largest proportion of ALE from the two granular sludges. In particular, the extracellular polymeric substances (EPS) extracted from bacterial and algal-bacterial AGS contained about 25.10 ± 1.85 and 19.53 ± 0.04 mg-P/g-SS, respectively, and both granular sludges possessed high P bioavailability of 97–99%.

Electrical and Electrochemical Characteristics of Withania somnifera Leaf Extract Incorporation Sodium Alginate Polymer Film for Energy Storage Applications

The current investigation addresses the alternating current electrical properties, electrochemical characteristics on biopolymer as Withania somnifera leaf extract incorporated sodium alginate [WLISA] polymer film prepared by solution casting method. Some crystallinity along with the amorphous nature of sodium alginate emerged due to the incorporation of Withania somnifera leaf extract [WSLE]. Two absorption maximum (λmax) were obtained for both WLISA and WSLE samples in the UV–Vis spectrum. The blue, green, and red emission of WSLE and weak blue emission of WLISA are studied by PL analysis. The more amount of strain that occurred in crystallization provides broaden peak in X-ray diffraction pattern. With aid of guluronic and mannuronic acids in sodium alginate making hydroxyl bond formation with leaf extract due to the cross-linking process can be analyzed from Fourier transform infrared spectroscopy. The EDAX analysis showed the chemical composition of WSLE. At room temperature, the prepared 5 ml leaf extract WLISA film has demonstrated the highest ionic conductivity in the typical value of 2.80 × 10–6 S/Cm. The electrochemical window of the prepared polymer film is in the range of − 0.4 to 0.4 V suggesting that used as an electrolyte for supercapacitor applications.

Bio-sourced polymers in cosmetic emulsions: a hidden potential of the alginates as thickeners and gelling agents.

The present work aims to investigate the impact of the alginates on the texture properties of cosmetic emulsions. For this purpose, five systems were selected: a classical emulsion without polymer and four emulsions containing polymers, as texture modifiers, at the level of 1%. Two different grades of alginates were chosen: one rich in mannuronic acid and one rich in guluronic acid. The objective was also to evaluate the potential of in-situ gelation during formulation. The guluronic rich sample was gelled to evaluate the effect on the texture properties. Finally, alginates-based systems were compared to the xanthan gum as a bio-sourced polymer reference. The sensory profile of the systems was established through a combination of prediction models and sensory analysis. The emulsion residual films obtained with natural polymers, Alginates and Xanthan Gum used as thickeners, as well as with the gelled version, were similar. However, the structural differences between polymers intervene during the characterisation of the sensory properties "before" and "during" application. A multi-scale physicochemical analysis was used to explain these differences. Due to a controlled formulation process, the use of the polymers did not affect the microstructure of the emulsion which remained similar to the control one. The main impact of the polymers was observed on the macroscopic level: both alginates showed their unique textural signature, different from the classical Xanthan Gum. Due to weak structural differences, mechanical and textural properties were very close between the mannuronic rich and guluronic rich samples, when not gelled, compared to other emulsions. However, the molar mass and the mannuronic/guluronic acids ratio were proved to be crucial for the stretching and consistency properties, showing that this structural difference may have an impact when products are handled in traction and compression. Meanwhile, the viscoelastic properties and the dynamic viscosity were greatly increased for the emulsion containing the gelled version of the alginate when compared to the classical polymers. The emulsion was also more consistent as proved by the textural analysis, pointing at better stability and suspension potential of the gelled emulsion versus the classical one containing the usual natural thickening agents.

Evaluation of the Effect of Mannuronic Acid as a Novel NSAID With Immunosuppressive Properties on Expression of SOCS1, SOCS3, SHIP1, and TRAF6 Genes and Serum Levels of IL-6 and TNF-α in Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is described as a chronic inflammatory, demyelinating disease of the central nervous system on an autoimmune basis, which is the most frequent reason for nontraumatic disability in youth. The efficacy and safety of β-D-nannuronic acid (M2000) as a novel immunosuppressive drug (patented PCT/EP2017/067920) has been shown in an experimental model of MS and also in a phase 2 clinical trial. The effects of M2000 on SOCS1, SOCS3, TRAF6, and SHIP1 gene expression and also serum levels of IL-6 and TNF-α in secondary progressive multiple sclerosis patients have been assessed in this study. In this study, 14 secondary progressive multiple sclerosis patients and 14 healthy subjects (as the control group) were recruited from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). Gene expression of SOCS1, SOCS3, TRAF6, and SHIP1 was measured at baseline and after 6months of therapy with M2000 using a quantitative real-time polymerase chain reaction method. Furthermore, the serum levels of IL-6 and TNF-α were assessed by the enzyme-linked immunosorbent assay method. Our results showed that the gene expression of SOCS1, SOCS3, and SHIP1 was increased after 6months of therapy with M2000 in MS patients. Moreover, the serum levels of IL-6 and TNF-α of patients declined compared with baseline, but this was not statistically significant. The results of this study demonstrated that M2000, with immunosuppressive properties, could upregulate SOCS1, SOCS3, and SHIP1 genes in patients with secondary progressive multiple sclerosis.

Chemical synthesis of C6-tetrazole ᴅ-mannose building blocks and access to a bioisostere of mannuronic acid 1-phosphate.

Alginate is a biocompatible and industrially relevant polysaccharide that derives many of its important properties from the charged carboxylate groups within its polyuronic acid backbone. The design and inclusion of isosteric replacements for these carboxylates would underpin provision of new oligo-/polysaccharide materials with alternate physicochemical properties. Presented herein is our synthesis of mannuronic acid building blocks, appropriately modified at the carboxylate C6 position with a bioisosteric tetrazole. Thioglycosides containing a protected C6-tetrazole are accessed from a C6-nitrile, through dipolar cycloaddition using NaN3 with n-Bu2SnO. We also demonstrate access to orthogonally C4-protected donors, suitable for iterative oligosaccharide synthesis. The development of these building blocks is showcased to access anomeric 3-aminopropyl- and 1-phosphate free sugars containing this non-native motif.

Production, structural and biochemical characterization relevant to antitumor property of acidic exopolysaccharide produced from Bacillus sp. NRC5

This study targeted the production of exopolysaccharide from Bacillus sp. NRC5 grown in Egyptian seawater to use it as natural antitumor therapy. The biological activities of selected exopolysaccharide (BEPS) as an antioxidant, anti-inflammatory and anticancer have been studied. BEPS was evaluated as an anti-inflammatory in vitro against cyclooxygenase (COX-1 and COX-2) and evaluated as an anticancer on human breast and prostate cancer cell lines (MCF-7 and PC3). In addition, BEPS antitumor activity was tested against the Ehrlich Ascites Carcinoma (EAC) model. The BEPS presented potent antioxidant activities, consisted of glucose, mannose, and mannuronic acid in a molar ratio of 1.0:1.7:0.8 with a molecular weight of 3.59 × 105g/mol. BEPS showed a promising COX-2 inhibitory effect in comparison with the reference drug celecoxib. BEPS appeared efficient anticancer property, where it killed 64.20 and 70.20% of MCF-7 and PC3 cells at 100µg/ml, respectively (IC50, 76.70, and 70.40µg/ml, respectively). BEPS exhibited antitumor ability as it prolonged the lifespan of mice to reach 75days instead of 20days in the tumor control, reduced viable cancer cells count, tumor volume and weight, modulated blood components, and white blood cells differentiation. BEPS produced from Bacillus sp. NRC5 showed its antioxidant and anti-inflammatory abilities and antitumor abilities, which may all be attributed to its unique composition containing sulfated moieties and uronic acids.

The influence of Na/H exchange on the charge transfer dissociation (CTD) spectra of mannuronic acid oligomers

This work focuses on the application of charge transfer dissociation (CTD) to a mannuronic acid oligomer with different extents of Na/H exchange. The goals were to determine if CTD could effectively characterize the oligomer and to investigate how Na/H exchanges impact the CTD fragmentation pathways of even a simple oligomer like mannuronic acid. CTD provided unique and unambiguous fragments that helped determine the 1-4-linkage pattern between core mannuronic acid residues. CTD of precursors with no Na/H exchange mainly had unambiguous product ions. However, CTD of precursor ions containing two or more Na/H exchanges increased the number of possible product ion identities and yielded many isobaric product ions and, hence, more ambiguity in spectral assignments. The use of 18O labelling on the reducing terminus helped resolve some isobaric interferences between A/X, and C/Z ions but created some nominal overlap with fragment ions containing Na/H exchanges. CTD spectra demonstrated that when three or more Na/H exchanges are present in the precursor, there are at least two main structures of Na/H exchange sites: the first structure contains three Na/H exchanges on adjacent sugars closest to the non-reducing terminus and a second structure contains two adjacent Na/H exchanges on the reducing terminus. When four or five Na/H exchanges are present, the positions of the fourth and fifth sodium ions could not be confirmed, presumably because the isolated precursors contain a distribution of structures with Na/H exchanges in different positions. In the absence of Na/H exchanges, He-CTD spectra provided rich structural information and very few ambiguous product ions. Therefore, until CTD is coupled to high-resolution mass spectrometers—which could readily resolve the ambiguity in most of the peak identities—precursors that contain the fewest possible number of Na/H exchanges will provide the most confident peak assignments and structural characterization.

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.

Controlled Delivery of Slit3 Proteins from Alginate Microbeads Inhibits In Vitro Angiogenesis

BackgroundThe Slit-Robo pathway is a key regulator of angiogenesis and cellular function in experimental models. Slit3 proteins exhibit both proangiogenic and antiangiogenic properties, but the exact mechanism remains unclear. It is theorized that Slit3 may be a potential treatment for vascular diseases and cancer. MethodsSlit3 labeled with I-125 was encapsulated in microbeads composed of low-viscosity alginate of high-glucuronic acid content, first coated with poly-L-ornithine for various durations and finally with low-viscosity high mannuronic acid. Gamma counter was used to measure microbead encapsulation efficiency and Slit3 release. Markers of angiogenesis were assessed with Boyden chamber, scratch wound, and Matrigel tube formation assays using human umbilical vein and mouse endothelial cells. ResultsOn incubation of Slit3-loaded microbeads, there was an initial burst phase release of Slit3 for the first 24 h followed by sustained release for 6 to 12 d. Microbead composition determined encapsulation efficiency and rate of release; Slit3 encapsulation was most efficient in microbeads with lower low-viscosity alginate of high-glucuronic acid content concentrations (1.5%) and no poly-L-ornithine coating. Compared with controls (media alone), Slit3 microbeads significantly inhibited in vitro cellular migration, endothelial cell migration for wound closure at 24 and 48 h and endothelial tube formation (P < 0.001, respectively). ConclusionsSlit3 can be effectively encapsulated and delivered via a controlled release pattern using alginate microbeads. Microbead encapsulation reduces in vitro endothelial tube formation and inhibits cellular migration to impair angiogenesis. Thus, Slit3 microparticles may be explored as a therapeutic option to mitigate tumor proliferation.

Cytosolic delivery of the immunological adjuvant Poly I:C and cytotoxic drug crystals via a carrier-free strategy significantly amplifies immune response

Co-delivery of chemotherapeutics and immunostimulant or chemoimmunotherapy is an emerging strategy in cancer therapy. The precise control of the targeting and release of agents is critical in this methodology. This article proposes the asynchronous release of the chemotherapeutic agents and immunostimulants to realize the synergistic effect between chemotherapy and immunotherapy. To obtain a proof-of-concept, a co-delivery system was prepared via a drug-delivering-drug (DDD) strategy for cytosolic co-delivery of Poly I:C, a synthetic dsRNA analog to activate RIG-I signaling, and PTX, a commonly used chemotherapeutics, in which pure PTX nanorods were sequentially coated with Poly I:C and mannuronic acid via stimulating the RIG-I signaling axis. The co-delivery system with a diameter of 200 nm enables profound immunogenicity of cancer cells, exhibiting increased secretion of cytokines and chemokines, pronounced immune response in vivo, and significant inhibition of tumor growth. Also, we found that intracellularly sustained release of cytotoxic agents could elicit the immunogenicity of cancer cells. Overall, the intracellular asynchronous release of chemotherapeutics and immunomodulators is a promising strategy to promote the immunogenicity of cancer cells and augment the antitumor immune response.

Influence of preservation methods on biochemical composition and downstream processing of cultivated Saccharina latissima biomass

Saccharina latissima biomass cultivated along the Swedish west coast was subjected to four different scalable preservation methods after harvest; freezing, sun-drying, oven-drying and ensiling. Freeze-drying and freezing at −80 °C were also included to provide dry and wet references. The effects of the different preservation methods on the composition of Saccharina biomass (on dry weight, DW, basis), and the recovery as well as properties of high-quality protein, alginate and biogas were evaluated. Sun-drying significantly reduced protein, alginate and fatty acid content of the seaweeds and thereby concentrated ash in the biomass compared to the other methods. Protein/amino acids and fatty acids were significantly concentrated in ensiled biomass, while mannitol and laminarin were reduced compared to the other biomasses. Oven-drying and −20 °C freezing affected the composition the least, with lower ash content and alterations in some specific amino and fatty acids. Sun-drying and ensiling resulted in significantly lower protein solubility at high pH compared to the other biomasses which translated into the lowest total seaweed protein recovery using the pH-shift process. Highest protein yield was obtained with the freeze-dried reference. Ensiling lead to a significant decrease in the molecular weight of alginate, while sun-drying caused a negative effect on alginate by inducing a shift in the guluronic and mannuronic acids composition of alginate. Sun-drying gave the lowest methane yield in the anaerobic digestion experiments while freezing at −80 °C gave the highest yield, closely followed by freezing at −20 °C and ensiling. To conclude, preservation methods must be carefully chosen to protect the valuable component in Saccharina latissima, and to achieve an efficient downstream processing ultimately yielding high quality products as part of a seaweed biorefinery.

Overexpression of Thermal and pH Stable Alginate Lyase of P. aeruginosa 293 and In silico Study of algL Gene

P. aeruginosa is an opportunistic bacterium that produces a capsule-like polysaccharide called alginate in response to various stimuli. The mucoid strain of Pseudomonas aeruginosa produces alginate which is an exopolysaccharide and is involved in the pathogenicity and persistence of these bacteria in infections. The alginate lyase gene is required for alginate synthesis. The enzyme can also degrade this polymer. This enzyme has a polymorphism in different bacteria even in one species and finding an enzyme with tremendous characters is very important. In this study, alginate lyase from P. aeruginosa strain 293 which was previously isolated from the sputum, and the encoding gene was characterized, and thermal and pH stability, as well as the substrate specificity of the partially purified alginate lyase, were determined. The amount of 70% activity of the enzyme was maintained after incubation at 80 ˚C for 6 hrs and 50% activity retained after incubation in alkaline and acidic pH. Moreover, it showed activity towards guluronic acid blocks, mannuronic acid blocks, and alginate blocks with both of them. Due to the unique properties of the alginate lyase that are useful in medicinal, and industrial applications, the gene encoding the enzyme was expressed in pET-28a (+)/E. coli BL21 (DE3) system to produce 371 -amino acid alginate lyase protein, the molecular weight of which was estimated by Sodium Dodecyl Sulfate- Polyacrylamide gel electrophoresis to be about 40 kDa. Bioinformatic analysis of P. aeruginosa strain 293 algL gene revealed that G225A point mutation can improve its thermostability. Therefore, the P. aeruginosa 293 alginate lyase is proposed as an appropriate candidate for the evaluation of potential therapeutic and industrial applications.

Functionalization of an Alginate-Based Material by Oxidation and Reductive Amination

This research focused on the synthesis of a functional alginate-based material via chemical modification processes with two steps: oxidation and reductive amination. In previous alginate functionalization with a target molecule such as cysteine, the starting material was purified and characterized by UV-Vis, 1H-NMR and HSQC. Additionally, the application of FT-IR techniques during each step of alginate functionalization was very useful, since new bands and spiked signals around the pyranose ring (1200–1000 cm−1) and anomeric region (1000–750 cm−1) region were identified by a second derivative. Additionally, the presence of C1-H1 of β-D-mannuronic acid residue as well as C1-H1 of α-L-guluronic acid residue was observed in the FT-IR spectra, including a band at 858 cm−1 with characteristics of the N-H moiety from cysteine. The possibility of attaching cysteine molecules to an alginate backbone by oxidation and post-reductive amination processes was confirmed through 13C-NMR in solid state; a new peak at 99.2 ppm was observed, owing to a hemiacetal group formed in oxidation alginate. Further, the peak at 31.2 ppm demonstrates the presence of carbon -CH2-SH in functionalized alginate—clear evidence that cysteine was successfully attached to the alginate backbone, with 185 μmol of thiol groups per gram polymer estimated in alginate-based material by UV-Visible. Finally, it was observed that guluronic acid residue of alginate are preferentially more affected than mannuronic acid residue in the functionalization.