RESUMEN

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 ß-elimination reaction, yielding oligosaccharides with an unsaturated sugar (4-deoxy-l-erythro-hex-4-enepyranosyluronate) at the non-reducing end. In this work we have systematically studied acid degradation of unsaturated uronic acid oligosaccharides. Based on these findings, a method for preparing saturated oligosaccharides by enzymatic degradation of uronic acid-containing polysaccharides was developed. This results in oligosaccharides with a pre-defined distribution and proportion of sugar residues compared to the products of chemical degradation, while maintaining the chemical structure of the non-reducing end. The described method was demonstrated for generating saturated oligosaccharides of alginate, heparin and polygalacturonic acid. In the case of alginate, the ratio of hydrolysis rate of Δ-G and Δ-M linkages to that of G-G and M-M linkages, respectively, was found to be approximately 65 and 43, at pH* 3.4, 90 °C. Finally, this method has been demonstrated to be superior in the production of α-l-guluronate oligosaccharides with a lower content of ß-d-mannuronate residues compared to what can be achieved using chemical depolymerization alone.

Asunto(s)

RESUMEN

Low molecular weight alginate oligosaccharides have been shown to exhibit anti-microbial activity against a range of multi-drug resistant bacteria, including Pseudomonas aeruginosa. Previous studies suggested that the disruption of calcium (Ca2+)-DNA binding within bacterial biofilms and dysregulation of quorum sensing (QS) were key factors in these observed effects. To further investigate the contribution of Ca2+ binding, G-block (OligoG) and M-block alginate oligosaccharides (OligoM) with comparable average size DPn 19 but contrasting Ca2+ binding properties were prepared. Fourier-transform infrared spectroscopy demonstrated prolonged binding of alginate oligosaccharides to the pseudomonal cell membrane even after hydrodynamic shear treatment. Molecular dynamics simulations and isothermal titration calorimetry revealed that OligoG exhibited stronger interactions with bacterial LPS than OligoM, although this difference was not mirrored by differential reductions in bacterial growth. While confocal laser scanning microscopy showed that both agents demonstrated similar dose-dependent reductions in biofilm formation, OligoG exhibited a stronger QS inhibitory effect and increased potentiation of the antibiotic azithromycin in minimum inhibitory concentration and biofilm assays. This study demonstrates that the anti-microbial effects of alginate oligosaccharides are not purely influenced by Ca2+-dependent processes but also by electrostatic interactions that are common to both G-block and M-block structures.

Asunto(s)

RESUMEN

Alginates are linear polysaccharides produced by brown algae and some bacteria and are composed of ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Alginate has numerous present and potential future applications within industrial, medical and pharmaceutical areas and G rich alginates are traditionally most valuable and frequently used due to their gelling and viscosifying properties. Mannuronan C-5 epimerases are enzymes converting M to G at the polymer level during the biosynthesis of alginate. The Azotobacter vinelandii epimerases AlgE1-AlgE7 share a common structure, containing one or two catalytic A-modules (A), and one to seven regulatory R-modules (R). Despite the structural similarity of the epimerases, they create different M-G patterns in the alginate; AlgE4 (AR) creates strictly alternating MG structures whereas AlgE1 (ARRRAR) and AlgE6 (ARRR) create predominantly G-blocks. These enzymes are therefore promising tools for producing in vitro tailor-made alginates. Efficient in vitro epimerization of alginates requires availability of recombinantly produced alginate epimerases, and for this purpose the methylotrophic yeast Hansenula polymorpha is an attractive host organism. The present study investigates whether H. polymorpha is a suitable expression system for future large-scale production of AlgE1, AlgE4, and AlgE6. H. polymorpha expression strains were constructed using synthetic genes with reduced repetitive sequences as well as optimized codon usage. High cell density cultivations revealed that the largest epimerases AlgE1 (147 kDa) and AlgE6 (90 kDa) are subject to proteolytic degradation by proteases secreted by the yeast cells. However, degradation could be controlled to a large extent either by co-expression of chaperones or by adjusting cultivation conditions. The smaller AlgE4 (58 kDa) was stable under all tested conditions. The results obtained thus point toward a future potential for using H. polymorpha in industrial production of mannuronan C-5 epimerases for in vitro tailoring of alginates.

RESUMEN

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 Azotobacter vinelandii 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 (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (1H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Asunto(s)

RESUMEN

Alginate is a linear copolymer composed of 1→4 linked ß-d-mannuronic acid (M) and its epimer α-l-guluronic acid (G). The polysaccharide is first produced as homopolymeric mannuronan and subsequently, at the polymer level, C-5 epimerases convert M residues to G residues. The bacterium Azotobacter vinelandii encodes a family of seven secreted and calcium ion-dependent mannuronan C-5 epimerases (AlgE1-AlgE7). These epimerases consist of two types of structural modules: the A-modules, which contain the catalytic site, and the R-modules, which influence activity through substrate and calcium binding. In this study, we rationally designed new hybrid mannuronan C-5 epimerases constituting the A-module from AlgE6 and the R-module from AlgE4. This led to a better understanding of the molecular mechanism determining differences in MG- and GG-block-forming properties of the enzymes. A long loop with either tyrosine or phenylalanine extruding from the ß-helix of the enzyme proved essential in defining the final alginate block structure, probably by affecting substrate binding. Normal mode analysis of the A-module from AlgE6 supports the results.

RESUMEN

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. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young's modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.

Asunto(s)

RESUMEN

In bacteria from the phylum Bacteroidetes, the genes coding for enzymes involved in polysaccharide degradation are often colocalized and coregulated in so-called "polysaccharide utilization loci" (PULs). PULs dedicated to the degradation of marine polysaccharides (e.g. laminaran, ulvan, alginate and porphyran) have been characterized in marine bacteria. Interestingly, the gut microbiome of Japanese individuals acquired, by lateral transfer from marine bacteria, the genes involved in the breakdown of porphyran, the cell wall polysaccharide of the red seaweed used in maki. Sequence similarity analyses predict that the human gut microbiome also encodes enzymes for the degradation of alginate, the main cell wall polysaccharide of brown algae. We undertook the functional characterization of diverse polysaccharide lyases from family PL17, frequently found in marine bacteria as well as those of human gut bacteria. We demonstrate here that this family is polyspecific. Our phylogenetic analysis of family PL17 reveals that all alginate lyases, which have all the same specificity and mode of action, cluster together in a very distinct subfamily. The alginate lyases found in human gut bacteria group together in a single clade which is rooted deeply in the PL17 tree. These enzymes were found in PULs containing PL6 enzymes, which also clustered together in the phylogenetic tree of PL6. Together, biochemical and bioinformatics analyses suggest that acquisition of this system appears ancient and, because only traces of two successful transfers were detected upon inspection of PL6 and PL17 families, the pace of acquisition of marine polysaccharide degradation system is probably very slow.

Asunto(s)

RESUMEN

The structure of fucoidan isolated from Laminaria hyperborea was elucidated and chemically tailored in order to obtain a clear structure-function relationship on bioactive properties with a minimal amount of variations among the tested molecules. Analysis revealed a sugar composition of 97.8% fucose and 2.2% galactose. Analysis of the glycosidic linkages showed (1→3)-α-l-fuco-pyranose (31.9%) to be the dominant residue, followed by 1→2-linked (13.2%) and 1→4-linked (7.7%) fuco-pyranose as well as a high degree of branching (22.4%). Inductively coupled plasma mass spectrometry (ICP-MS) revealed a sulfate content of 53.8% (degree of sulfation (DS) = 1.7). Raman spectroscopy determined SO4 located axial at 4C and equatorial at 2C as well as an absence of acetylation. SEC-MALS analysis determined a high molecular weight (Mw = 469 kDa), suggesting a highly flexible main chain with short side chains. Both chemical shifts of the fucoidan, proton, and carbon were assigned by NMR and revealed a highly heterogeneous structure in terms of glycosidic linkages. Bioactivity was assessed using a lepirudin-based whole blood model. The immediate responses by coagulation and complement cascades were measured by prothrombine factor 1 and 2 (PTF1.2) and the terminal complement complex (TCC). Cytokines involved in inflammation were detected in a 27-plex cytokine assay. Fucoidan with a high Mw and DS inhibited coagulation, complement, and the cytokines PDGF-BB, RANTES, and IP-10, while activating MCP-1. These effects were obtained at the concentration of 1000 ug/mL and partly at 100 ug/mL. In low concentrations (10 ug/mL), a coagulation stimulating effect of highly sulfated fucoidans (DS = 1.7, Mw = 469 kDa or 20.3) was obtained. These data point to a multitude of effects linked to the sulfation degree that needs further mechanistic exploration.

RESUMEN

A family of seven mannuronan C5-epimerases (AlgE1-AlgE7) produced by Azotobacter vinelandii is able to convert ß-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. However, epimerization is hampered when the substrate is modified or in the gelled state. Here it is presented how native and engineered epimerases of varying size perform on steric hindered alginate substrates (modified or as hydrogels). Reducing the size of the epimerases enables the epimerization of otherwise inaccessible regions in the alginate polymer. Even though the reduction of the size affects the productive binding of epimerases to the substrate, and hence their activity, the smaller epimerases could more freely diffuse into calcium-alginate hydrogel and epimerize it.

Asunto(s)

RESUMEN

We evaluate an AFM-based single molecule force spectroscopy method for mapping sequences in otherwise difficult to sequence heteropolymers, including glycosylated proteins and glycans. The sliding contact force spectroscopy (SCFS) method exploits a sliding contact made between a nanopore threaded over a polymer axle and an AFM probe. We find that for sliding α- and ß-cyclodextrin nanopores over a wide range of hydrophilic monomers, the free energy of sliding is proportional to the sum of two dimensionless, easily calculable parameters representing the relative partitioning of the monomer inside the nanopore or in the aqueous phase, and the friction arising from sliding the nanopore over the monomer. Using this relationship we calculate sliding energies for nucleic acids, amino acids, glycan and synthetic monomers and predict on the basis of these calculations that SCFS will detect N- and O-glycosylation of proteins and patterns of sidechains in glycans. For these applications, SCFS offers an alternative to sequence mapping by mass spectrometry or newly-emerging nanopore technologies that may be easily implemented using a standard AFM.

RESUMEN

Heparin is widely recognized for its potent anticoagulating effects, but has an additional wide range of biological properties due to its high negative charge and heterogeneous molecular structure. This heterogeneity has been one of the factors in motivating the exploration of functional analogues with a more predictable modification pattern and monosaccharide sequence, that can aid in elucidating structure-function relationships and further be structurally customized to fine-tune physical and biological properties toward novel therapeutic applications and biomaterials. Alginates have been of great interest in biomedicine due to their inherent biocompatibility, gentle gelling conditions, and structural versatility from chemo-enzymatic engineering, but display limited interactions with cells and biomolecules that are characteristic of heparin and the other glycosaminoglycans (GAGs) of the extracellular environment. Here, we review the chemistry and physical and biological properties of sulfated alginates as structural and functional heparin analogues, and discuss how they may be utilized in applications where the use of heparin and other sulfated GAGs is challenging and limited.

Asunto(s)

RESUMEN

The goal of this study was to determine whether the guluronate (G) rich alginate OligoG CF-5/20 (OligoG) could detach cystic fibrosis (CF) mucus by calcium chelation, which is also required for normal mucin unfolding. Since bicarbonate secretion is impaired in CF, leading to insufficient mucin unfolding and thereby attached mucus, and since bicarbonate has the ability to bind calcium, we hypothesized that the calcium chelating property of OligoG would lead to detachment of CF mucus. Indeed, OligoG could compete with the N-terminus of the MUC2 mucin for calcium binding as shown by microscale thermophoresis. Further, effects on mucus thickness and attachment induced by OligoG and other alginate fractions of different length and composition were evaluated in explants of CF mouse ileum mounted in horizontal Ussing-type chambers. OligoG at 1.5% caused effective detachment of CF mucus and the most potent alginate fraction tested, the poly-G fraction of about 12 residues, had similar potency compared to OligoG whereas mannuronate-rich (M) polymers had minimal effect. In conclusion, OligoG binds calcium with appropriate affinity without any overt harmful effect on the tissue and can be exploited for treating mucus stagnation.

Asunto(s)

RESUMEN

Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encapsulated insulin-producing tissue is briefly discussed. Stem Cells Translational Medicine 2017;6:1053-1058.

Asunto(s)

RESUMEN

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. In addition, it permits bond rupture measurements in systems where one of the binding partners may be introduced into solution prior to binding without tethering to a surface. Taking as an example of a slowly forming bond, the "eggbox" junction cross-links between oligoguluronic acid chains (oligoGs) in the commercially important polysaccharide alginate, we show that SCFS accurately measures the equilibrated bond strength of the cross-link when one chain is introduced into the sample solution without tethering to a surface. The results validate the SCFS technique for performing single-molecule force spectroscopy experiments and show that it has advantages in cases where the bond to be studied forms slowly and where tethering of one of the binding partners is impractical.

RESUMEN

Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate lyases define a class of enzymes that cleave the glycosidic bond of alginate by ß-elimination. They are classified according to their ability to recognize the distribution of M- and G-residues and are named M-, G- or MG-lyases. In the CAZy database, alginate lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate lyases and one dermatan sulfatase lyase). No characterized enzymes have been described in the two other subfamilies (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we re-examined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates lyases and several alginate lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-lyases.

Asunto(s)

RESUMEN

UNLABELLED: Alginate microspheres show promise for cell-encapsulation therapy but encounter challenges related to biocompatibility. In the present work we designed novel microbeads and microcapsules based on sulfated polyalternating MG alginate (SMG) and explored their inflammatory properties using a human whole blood model. SMG was either incorporated within the alginate microbeads or used as a secondary coat on poly-l-lysine (PLL)-containing microcapsules, resulting in reduction of the inflammatory cytokines (IL-1ß, TNF, IL-6, IL-8, MIP-1α). The sulfated alginate microbeads exhibited a complement inert nature with no induction of terminal complement complex (TCC) above the values in freshly drawn blood and low surface accumulation of C3/C3b/iC3b. Conversely, SMG as a coating material lead to substantial TCC amounts and surface C3/C3b/iC3b. A common thread was an increased association of the complement inhibitor factor H to the alginate microbeads and microcapsules containing sulfated alginates. Factor H was also found to associate to non-sulfated alginate microbeads in lower amounts, indicating factor H binding as an inherent property of alginate. We conclude that the dampening effect on the cytokine response and increased factor H association points to sulfated alginate as a promising strategy for improving the biocompatibility of alginate microspheres. STATEMENT OF SIGNIFICANCE: Alginate microspheres are candidate devices for cell encapsulation therapy. The concept is challenged by the inflammatory host response, and modification strategies for improved biocompatibility are urgently needed. One potential strategy is using sulfated alginates, acting as versatile heparin analogues with similar anti-inflammatory properties. We designed novel alginate microspheres using sulfated alginate with an alternating sequence mimicking glycosominoglycans. Evaluation in a physiologically relevant human whole blood model revealed a reduction of inflammatory cytokines by a sulfated alginate coating, and sulfated alginate microbeads were complement inert. These effects were correlated with a strong factor H association, which may represent the mechanistic explanation. This novel approach could improve the biocompatibility of alginate microspheres in vivo and present a new strategy toward clinical use.

Asunto(s)

RESUMEN

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. Extending the interaction time from tens to hundreds of milliseconds allows longer oligoGs to achieve much stronger crosslinks but does not change the strength of individual links between shorter oligoGs. These results are considered in light of extant models for the onset of cooperative crosslinking in polyelectrolytes and an anisotropic distribution of oligoGs on interacting surfaces and provide a timescale for the formation and relaxation of alginate gels at the single crosslink level.

Asunto(s)

RESUMEN

The inflammatory potential of 12 types of alginate-based microspheres was assessed in a human whole blood model. The inflammatory potential could be categorized from low to high based on the four main alginate microsphere types; alginate microbeads, liquefied core poly-l-ornithine (PLO)-containing microcapsules, liquefied core poly-l-lysine (PLL)-containing microcapsules, and solid core PLL-containing microcapsules. No complement or inflammatory cytokine activation was detected for the Ca/Ba alginate microbeads. Liquefied core PLO- and PLL-containing microcapsules induced significant fluid phase complement activation (TCC), but with low complement surface deposition (anti-C3c), and a low proinflammatory cytokine secretion, with exception of an elevated MCP-1(CCL2) secretion. The solid core PLL-containing microcapsules generated lower TCC but a marked complement surface deposition and significant induction of the proinflammatory cytokines interleukin (IL-1)ß, TNF, IL-6, the chemokines IL-8 (CXCL8), and MIP-1α (CCL3) and MCP-1(CCL2). Inhibition with compstatin (C3 inhibitor) completely abolished complement surface deposition, leukocyte adhesion and the proinflammatory cytokines. The C5 inhibitions partly lead to a reduction of the proinflammatory cytokines. The leukocyte adhesion was abolished by inhibitory antibodies against CD18 and partly reduced by CD11b, but not by CD11c. Anti-CD18 significantly reduced the (IL-1)ß, TNF, IL-6 and MIP-1α and anti-CD11b significantly reduced the IL-6 and VEGF secretion. MCP-1 was strongly activated by anti-CD18 and anti-CD11b. In conclusion the initial proinflammatory cytokine responses are driven by the microspheres potential to trigger complement C3 (C3b/iC3b) deposition, leukocyte activation and binding through complement receptor CR3 (CD11b/CD18). MCP-1 is one exception dependent on the fluid phase complement activation mediated through CR3.

Asunto(s)

RESUMEN

Deciphering the roles of chemical and physical features of the extracellular matrix (ECM) is vital for developing biomimetic materials with desired cellular responses in regenerative medicine. Here, we demonstrate that sulfation of biopolymers, mimicking the proteoglycans in native tissues, induces mitogenicity, chondrogenic phenotype, and suppresses catabolic activity of chondrocytes, a cell type that resides in a highly sulfated tissue. We show through tunable modification of alginate that increased sulfation of the microenvironment promotes FGF signaling-mediated proliferation of chondrocytes in a three-dimensional (3D) matrix independent of stiffness, swelling, and porosity. Furthermore, we show for the first time that a biomimetic hydrogel acts as a 3D signaling matrix to mediate a heparan sulfate/heparin-like interaction between FGF and its receptor leading to signaling cascades inducing cell proliferation, cartilage matrix production, and suppression of de-differentiation markers. Collectively, this study reveals important insights on mimicking the ECM to guide self-renewal of cells via manipulation of distinct signaling mechanisms.

RESUMEN

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase-polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xß), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase-alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.

Asunto(s)