A chemo-enzymatic method for preparation of saturated oligosaccharides from alginate and other uronic acid-containing polysaccharides.

Oligosaccharides from uronic acid-containing polysaccharides can be produced either by chemical or enzymatic degradation. The benefit of using enzymes, called lyases, is their high specificity for various glycosidic linkages. Lyases cleave the polysaccharide chain by an β-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end.

Hydrogels based on methylated-alginates as a platform to investigate the effect of material properties on cell activity. The role of material compliance.

Alginate-based hydrogels with tunable mechanical properties are developed by chemical methylation of the polysaccharide backbone, which was performed either in homogeneous phase (in solution) or in heterogeneous phase (on hydrogels). Nuclear Magnetic Resonance (NMR) and Size Exclusion Chromatography (SEC-MALS) analyses of methylated alginates allow to identify the presence and location of methyl groups on the polysaccharide, and to investigate the influence of methylation on the stiffness of the polymer chains. The methylated polysaccharides are employed for the manufacturing of calcium-reticulated hydrogels for cell growth in 3D.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.

Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production.

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (, and ) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of NCIMB 10,525.

Bi-Functional Alginate Oligosaccharide-Polymyxin Conjugates for Improved Treatment of Multidrug-Resistant Gram-Negative Bacterial Infections.

The recent emergence of resistance to colistin, an antibiotic of last resort with dose-limiting toxicity, has highlighted the need for alternative approaches to combat infection. This study aimed to generate and characterise alginate oligosaccharide ("OligoG")-polymyxin (polymyxin B and E (colistin)) conjugates to improve the effectiveness of these antibiotics. OligoG-polymyxin conjugates (amide- or ester-linked), with molecular weights of 5200-12,800 g/mol and antibiotic loading of 6.

Functional characterization of three Azotobacter chroococcum alginate-modifying enzymes related to the Azotobacter vinelandii AlgE mannuronan C-5-epimerase family.

Bacterial alginate initially consists of 1-4-linked β-D-mannuronic acid residues (M) which can be later epimerized to α-L-guluronic acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A.

Polymer Masked-Unmasked Protein Therapy: Identification of the Active Species after Amylase Activation of Dextrin-Colistin Conjugates.

Polymer masked-unmasked protein therapy (PUMPT) uses conjugation of a biodegradable polymer, such as dextrin, hyaluronic acid, or poly(l-glutamic acid), to mask a protein or peptide's activity; subsequent locally triggered degradation of the polymer at the target site regenerates bioactivity in a controllable fashion. Although the concept of PUMPT is well established, the relationship between protein unmasking and reinstatement of bioactivity is unclear. Here, we used dextrin-colistin conjugates to study the relationship between the molecular structure (degree of unmasking) and biological activity.

Biosynthesis and Function of Long Guluronic Acid-Blocks in Alginate Produced by Azotobacter vinelandii.

With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties.

Production, Characterization, and Application of an Alginate Lyase, AMOR_PL7A, from Hot Vents in the Arctic Mid-Ocean Ridge.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a β-d-mannuronate specific (EC 4.

Preparation of 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid (DEH) and Guluronic Acid Rich Alginate Using a Unique exo-Alginate Lyase from Thalassotalea crassostreae.

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. However, their commercial exploitation is currently limited by a lack of appropriate and efficient enzymes for converting alginate into metabolizable building blocks, such as 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein, we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-β-D-mannuronate (poly M) alginate, with a k of 135.

Overall size of mannuronan C5-Epimerases influences their ability to epimerize modified alginates and alginate gels.

A family of seven mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii is able to convert β-d-mannuronate (M) to its epimer α-l-guluronate (G) in alginates. Even sharing high sequence homology at the amino acid level, they produce distinctive epimerization patterns. The introduction of new G-blocks into the polymer by in vitro epimerization is a strategy to improve the mechanical properties of alginates as biomaterial.

Sliding Contact Dynamic Force Spectroscopy Method for Interrogating Slowly Forming Polymer Cross-Links.

Dynamic single-molecule force spectroscopy (SMFS), conducted most commonly using AFM, has become a widespread and valuable tool for understanding the kinetics and thermodynamics of fundamental molecular processes such as ligand-receptor interactions and protein unfolding. Where slowly forming bonds are responsible for the primary characteristics of a material, as is the case in cross-links in some polymer gels, care must be taken to ensure that a fully equilibrated bond has first formed before its rupture can be interpreted. Here we introduce a method, sliding contact force spectroscopy (SCFS), that effectively eliminates the kinetics of bond formation from the measurement of bond rupture.

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge.

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca).

Single molecule investigation of the onset and minimum size of the calcium-mediated junction zone in alginate.

One of the principal roles of alginate, both natively and in commercial applications, is gelation via Ca(2+)-mediated crosslinks between blocks of guluronic acid. In this work, single molecule measurements were carried out between well-characterised series of nearly monodisperse guluronic acid blocks ('oligoGs') using dynamic force spectroscopy. The measurements provide evidence that for interaction times on the order of tens of milliseconds the maximum crosslink strength is achieved by pairs of oligoGs long enough to allow the coordination of 4Ca(2+) ions, with both shorter and longer oligomers forming weaker links.

Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii.

The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module.

Lyase-catalyzed degradation of alginate in the gelled state: effect of gelling ions and lyase specificity.

Lyase-catalyzed degradation has been proposed as a more cell-friendly alternative to dissolution of alginate gels than using chelating agents. In this study, we investigated the effect of lyase specificity on degradation of alginate gels, including the effect of crosslinking ions with different affinity for the polymer. Degradation kinetics and products were analyzed.

Gelling concept combining chitosan and alginate-proof of principle.

Biocompatible hydrogels are very interesting for applications in, e.g., tissue engineering and for immobilization of cells, such as calcium-alginate gels where the calcium ions form specific interactions with the guluronic acid units.

Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.

Distribution and proportion of β-D-mannuronic and α-L-guluronic acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks).

Isolation of mutant alginate lyases with cleavage specificity for di-guluronic acid linkages.

Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1-4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction.

Preparation of high purity monodisperse oligosaccharides derived from mannuronan by size-exclusion chromatography followed by semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection.

Oligosaccharides of ([4)-beta-d-ManpA-(-->](n)) with a degree of polymerisation (DP) of 5, 10 and 15 were generated by partial acid hydrolysis of alginate mannuronan. These were subsequently purified by a combination of size-exclusion chromatography and semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. The purity of the isolated oligosaccharides was greater than 96%.

Application of high-performance anion-exchange chromatography with pulsed amperometric detection and statistical analysis to study oligosaccharide distributions--a complementary method to investigate the structure and some properties of alginates.

Alginates comprised of essentially alternating units of mannuronic (M) acid-guluronic (G) acid (MG-alginate), and G-blocks isolated from a seaweed where subjected to partial acid hydrolysis at pH 3.5 The chain-length distribution of oligosaccharides in the hydrolysate were investigated by statistical analysis after their separation with high-performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). Simulated depolymerisation of the MG-alginate provided an estimate of the ratio between two acid hydrolysis rate constants (p=8.