Association with Imidazole in the Cooperative Order-Disorder Transition in Aqueous Solution of Schizophyllan.

Schizophyllan, a triple helical polysaccharide, exhibits cooperative order-disorder transition (CODT) in aqueous solutions. The transition transforms the ordered structure (triple helix I) formed between the branched side chains and solvent molecules into the disordered structure (triple helix II) without dissociation of the triple helix. The CODT behaviors in H2O-imidazole mixtures containing HCl with different molar ratios of imidazole/HCl were investigated by adiabatic calorimetry and differential scanning calorimetry on two schizophyllan solutions with different molar masses. The transition temperature (Tr) and the transition enthalpy (ΔHr) significantly depended on both of the mole fractions of imidazole and imidazole/HCl. The composition dependences of Tr and ΔHr in H2O-imidazole mixtures were analyzed with linear cooperative transition theory for the solvent-stabilizing effect in the mixture with active compounds. Theoretical analyses confirmed that both imidazole and imidazolium ions in the solutions competitively interact with the side chain of the triple helix.

Structural and functional aspects of mannuronic acid-specific PL6 alginate lyase from the human gut microbe Bacteroides cellulosilyticus.

Alginate is a linear polysaccharide from brown algae consisting of 1,4-linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) arranged in M, G, and mixed MG blocks. Alginate was assumed to be indigestible in humans, but bacteria isolated from fecal samples can utilize alginate. Moreover, genomes of some human gut microbiome-associated bacteria encode putative alginate-degrading enzymes. Here, we genome-mined a polysaccharide lyase family 6 alginate lyase from the gut bacterium Bacteroides cellulosilyticus (BcelPL6). The structure of recombinant BcelPL6 was solved by X-ray crystallography to 1.3 Å resolution, revealing a single-domain, monomeric parallel ß-helix containing a 10-step asparagine ladder characteristic of alginate-converting parallel ß-helix enzymes. Substitutions of the conserved catalytic site residues Lys-249, Arg-270, and His-271 resulted in activity loss. However, imidazole restored the activity of BcelPL6-H271N to 2.5% that of the native enzyme. Molecular docking oriented tetra-mannuronic acid for syn attack correlated with M specificity. Using biochemical analyses, we found that BcelPL6 initially releases unsaturated oligosaccharides of a degree of polymerization of 2-7 from alginate and polyM, which were further degraded to di- and trisaccharides. Unlike other PL6 members, BcelPL6 had low activity on polyMG and none on polyG. Surprisingly, polyG increased BcelPL6 activity on alginate 7-fold. LC-electrospray ionization-MS quantification of products and lack of activity on NaBH4-reduced octa-mannuronic acid indicated that BcelPL6 is an endolyase that further degrades the oligosaccharide products with an intact reducing end. We anticipate that our results advance predictions of the specificity and mode of action of PL6 enzymes.

2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides.

Diblock oligosaccharides based on renewable resources allow for a range of new but, so far, little explored biomaterials. Coupling of blocks through their reducing ends ensures retention of many of their intrinsic properties that otherwise are perturbed in classical lateral modifications. Chitin is an abundant, biodegradable, bioactive, and self-assembling polysaccharide. However, most coupling protocols relevant for chitin blocks have shortcomings. Here we exploit the highly reactive 2,5-anhydro-d-mannose residue at the reducing end of chitin oligomers obtained by nitrous acid depolymerization. Subsequent activation by dihydrazides or dioxyamines provides precursors for chitin-based diblock oligosaccharides. These reactions are much faster than for other carbohydrates, and only acyclic imines (hydrazones or oximes) are formed (no cyclic N-glycosides). α-Picoline borane and cyanoborohydride are effective reductants of imines, but in contrast to most other carbohydrates, they are not selective for the imines in the present case. This could be circumvented by a simple two-step procedure. Attachment of a second block to hydrazide- or aminooxy-functionalized chitin oligomers turned out to be even faster than the attachment of the first block. The study provides simple protocols for the preparation of chitin-b-chitin and chitin-b-dextran diblock oligosaccharides without involving protection/deprotection strategies.

Conformation and cooperative order-disorder transition in aqueous solutions of ß-1,3-d-glucan with different degree of branching varied by the Smith degradation.

ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.

Influence of amino acids, buffers, and ph on the γ-irradiation-induced degradation of alginates.

Alginate-based biomaterials and medical devices are commonly subjected to γ-irradiation as a means of sterilization, either in the dry state or the gel (hydrated) state. In this process the alginate chains degrade randomly in a dose-dependent manner, altering alginates' material properties. The addition of free radical scavenging amino acids such as histidine and phenylalanine protects the alginate significantly against degradation, as shown by monitoring changes in the molecular weight distributions using SEC-MALLS and determining the pseudo first order rate constants of degradation. Tris buffer (0.5 M), but not acetate, citrate, or phosphate buffers had a similar effect on the degradation rate. Changes in pH itself had only marginal effects on the rate of alginate degradation and on the protective effect of amino acids. Contrary to previous reports, the chemical composition (M/G profile) of the alginates, including homopolymeric mannuronan, was unaltered following irradiation up to 10 kGy.

Alginate-metal cation interactions: Macromolecular approach.

Alginates are a broad family of linear (unbranched) polysaccharides derived from brown seaweeds and some bacteria. Despite having only two monomers, i.e. ß-d-mannuronate (M) and its C5 epimer α-l-guluronate (G), their blockwise arrangement in oligomannuronate (..MMM..), oligoguluronate (..GGG..), and polyalternating (..MGMG..) blocks endows it with a rather complex interaction pattern with specific counterions and salts. Classic polyelectrolyte theories well apply to alginate as polyanion in the interaction with monovalent and non-gelling divalent cations. The use of divalent gelling ions, such as Ca2+, Ba2+ or Sr2+, provides thermostable homogeneous or heterogeneous hydrogels where the block composition affects both macroscopic and microscopic properties. The mechanism of alginate gelation is still explained in terms of the original egg-box model, although over the years some novel insights have been proposed. In this review we summarize several decades of research related to structure-functionships in alginates in the presence of non-gelling and gelling cations and present some novel applications in the field of self-assembling nanoparticles and use of radionuclides.

Ionically gelled alginate foams: physical properties controlled by operational and macromolecular parameters.

Alginates in the format of scaffolds provide important functions as materials for cell encapsulation, drug delivery, tissue engineering and wound healing among others. The method for preparation of alginate-based foams presented here is based on homogeneous, ionotropic gelation of aerated alginate solutions, followed by air drying. The method allows higher flexibility and better control of the pore structure, hydration properties and mechanical integrity compared to foams prepared by other techniques. The main variables for tailoring hydrogel properties include operational parameters such as degree of aeration and mixing times and concentration of alginate, as well as macromolecular properties such as the type of alginate (chemical composition and molecular weight distribution). Exposure of foams to γ-irradiation resulted in a dose-dependent (0-30 kGy) reduction in molecular weight of the alginate and a corresponding reduction in tensile strength of the foams.

Dicarboxylated hyaluronate: Synthesis of a new, highly functionalized and biocompatible derivative.

Sequential periodate-chlorite oxidation of sodium hyaluronate to 2,3-dicarboxylated hyaluronate (DCH), a novel biocompatible and highly functionalized derivative bearing additional pair of COOH groups at C2 and C3 carbons of oxidized ᴅ-glucuronic acid units, is investigated. The impact of various reaction parameters (time, oxidizer concentration, and molar amount) on DCH's composition, molecular weight, degree of oxidation, and cytotoxicity are investigated to guide the synthesis of DCH derivatives of desired properties. Subsequently, fully (99%) and partially (70%) oxidized DCH derivatives were compared to untreated sodium hyaluronate in terms of anticancer drug cisplatin loading efficacy, carrier capacity, drug release rates, and cytotoxicity towards healthy and cancerous cell lines. DCH derivatives were found to be superior in every aspect, having nearly twice the carrier capacity, significantly slower release rates, and higher efficacy. DCH is thus a highly interesting hyaluronate derivative with an adjustable degree of oxidation, molecular weight, and great potential for further modifications.

Carbohydr Polym Special Issue Invited contribution: Click chemistry for block polysaccharides with dihydrazide and dioxyamine linkers - A review.

Engineered block polysaccharides is a relatively new class of biomacromolecules consisting of chemical assembly of separate block structures at the chain termini. In contrast to conventional, laterally substituted polysaccharide derivatives, the block arrangement allows for much higher preservation of inherent chain properties such as biodegradability and stimuli-responsive self-assembly, while at the same time inducing new macromolecular properties. Abundant, carbon neutral, and even recalcitrant biomass is an excellent source of blocks, opening for numerous new uses of biomass for a wide range of novel biomaterials. Among a limited range of methodologies available for block conjugation, bifunctional linkers allowing for oxyamine and hydrazide 'click' reactions have recently proven useful additions to the repertoire. This article focuses the chemistry and kinetics of these reactions. It also presents some new data with the aim to provide useful protocols and methods for general use towards new block polysaccharides.

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus.

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin-the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

Functionalisation of the non-reducing end of chitin by selective periodate oxidation: A new approach to form complex block polysaccharides and water-soluble chitin-based block polymers.

Most polysaccharides used in polysaccharide-based block copolymers are attached to the second block through the reducing end, due to the few and highly polysaccharide specific non-reducing end (NRE) functionalisation methods available. Chitin oligomers, prepared by nitrous acid degradation of chitosan (AnM) can, however, be selectively oxidised by periodate since they only possess a single vicinal diol in the NRE residue. Here, we show that both aldehydes formed after oxidation are highly reactive towards bifunctional oxyamines and hydrazide linkers. Sub-stochiometric amounts of linkers resulted in conjugation of AnM oligomers through both chain termini to yield a discrete distribution of 'polymerised' oligomers. Such chitin-based block polymers were, in contrast to chitins of the same chain lengths, water-soluble. Oxidised AnM oligomers, functionalised at both termini can also enable the preparation of more complex block polysaccharides such as ABA- or ABC-type.

Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides.

Reducing end activation of poly- and oligosaccharides by bifunctional dioxyamines and dihydrazides enables aniline-free and cyanoborohydride-free conjugation to aldehyde-containing molecules, particles and surfaces without compromising the chain structure. Chitosans are due to their polycationic character, biodegradability, and bioactivity important candidates for conjugation. Here, we present a kinetic and structural study of the conjugation of a dioxyamine and a dihydrazide to enzymatically produced chitooligosaccharides ranging from N,N'-diacetylchitobiose to a decamer, all having N-acetyl d-glucosamine at the reducing end. Conjugation of the dioxyamine resulted in mixtures of (E)- and (Z)-oximes and ß-N-pyranoside, whereas the dihydrazide yielded cyclic N-glycosides. Reaction kinetics was essentially independent of DP. Stable secondary amines were in both cases obtained by reduction with α-picoline borane, but higher temperatures were needed to obtain acceptable reduction rate. Comparison to dextran oligomers shows that the nature of the reducing end strongly influences the kinetics of both the conjugation and reduction.

Sterilization effects on the handling and degradation properties of calcium phosphate cements containing poly (D,L -lactic-co-glycolic acid) porogens and carboxymethyl cellulose.

Injectable, self-setting calcium phosphate cements (CPCs) are synthetic bone substitutes considered favorable for the repair and regeneration of bone due to their osteocompatibility and unique handling properties. However, their clinical applicability can be compromised due to insufficient cohesion upon injection into the body coupled with poor degradation rates that restricts new bone formation. Consequently, carboxymethyl cellulose (CMC) was incorporated into CPC formulations to improve their cohesion and injectability while poly (D,L -lactic-co-glycolic acid) (PLGA) porogens were added to introduce macroporosity and improve their biodegradation rate. Like most biomaterials, CPCs are gamma irradiated before clinical use to ensure sufficient sterilization. However, it is well known that gamma irradiation also reduces the molecular weight of CMC and PLGA via chain scission, which affects their material properties. Therefore, the aim of this study is to measure the effect that gamma irradiation has on the molecular weight of CMC at varying doses of 15, 40, or 80 kGy and investigate how this affects the handling (i.e., injectability, cohesion, washout, and setting times) and in vitro degradation behavior of CPC formulations. Results reveal that the molecular weight of CMC decreases with increasing gamma irradiation dose, thereby reducing the viscosifying capabilities of CMC, which causes CPCs to deteriorate more readily. Further, the addition of CMC seems to inhibit the degree of phase transformation during cement setting while the subsequent reduction in molecular weight of PLGA after gamma irradiation improves the in vitro degradation rate of CPCs due to the faster degradation rate of low molecular weight PLGA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2216-2228, 2019.

The Azotobacter vinelandii AlgE mannuronan C-5-epimerase family is essential for the in vivo control of alginate monomer composition and for functional cyst formation.

The industrially widely used polysaccharide alginate is a co-polymer of beta-D-mannuronic acid and alpha-L-guluronic acid (G), and the G residues originate from a polymer-level epimerization process catalysed by mannuronan C-5-epimerases. In the genome of the alginate-producing bacterium Azotobacter vinelandii genes encoding one periplasmic (AlgG) and seven secreted such epimerases (AlgE1-7) have been identified. Here we report the generation of a strain (MS163171) in which all the algE genes were inactivated by deletion (algE1-4 and algE6-7) or interruption (algE5). Shake flask-grown MS163171 produced a polymer containing less than 2% G (algG still active), while wild-type alginates contained 25% G. Interestingly, addition of proteases to the MS163171 growth medium resulted in a strong increase in the chain lengths of the alginates produced. MS163171 was found to be unable to form functional cysts, which is a desiccation-resistant differentiated form developed by A. vinelandii under certain environmental conditions. We also generated mutants carrying interruptions in each separate algE gene, and a strain containing algE5 only. Studies of these mutants indicated that single algE gene inactivations, with the exception of algE3, did not affect the fractional G content much. However, for all strains tested the alginate composition varied somewhat as a response to the growth conditions.

Tailoring of chitosans for gene delivery: novel self-branched glycosylated chitosan oligomers with improved functional properties.

Chitosan is a promising biomaterial with an attractive safety profile; however, its application potential for gene delivery is hampered by poor compatibility at physiological pH values. Here we have tailored the molecular architecture of chitosan to improve the functional properties and gene transfer efficacy of chitosan oligomers and have developed self-branched glycosylated chitosan oligomer (SB-TCO) substituted with a trisaccharide containing N-acetylglucosamine, AAM. SB-TCO was prepared by controlled depolymerization of chitosan, followed by simultaneous branching and AAM substitution. The product was fully soluble at physiological pH and complexed plasmid DNA into polyplexes of high colloidal and physical stability. SB-TCO displayed high transfection efficacy in HEK293 cells, reaching transfection efficiencies of up to 70%, and large amounts of transgene were produced. Gene transfer efficacy was confirmed in HepG2 cells, where gene expression levels mediated by SB-TCO were up to 10 and 4 times higher than those obtained with unsubstituted and substituted linear oligomers, respectively. The rapid onset of transgene expression in both cell lines indicates efficient DNA release and transcription from SB-TCO polyplexes. In comparison with 22 kDa linear PEI-based transfection reagent used as the control, SB-TCO possessed higher gene transfer efficacy, significantly lower cytotoxicity, and improved serum compatibility.

Cooperative Order-Disorder Transition of Carboxylated Schizophyllan in Water-Dimethylsulfoxide Mixtures.

Carboxylated schizophyllan ("sclerox") is a chemically modified polysaccharide obtained by partial periodate oxidation and subsequent chlorite oxidation of schizophyllan, a water-soluble neutral polysaccharide having a ß-1,3-linked glucan backbone and a ß-1,6-linked d-glucose residue side chain at every third residue of the main chain. The triple helix of schizophyllan in water has a cooperative order-disorder transition associated with the side chains. The transition is strongly affected by the presence (mole fraction) of dimethylsulfoxide (DMSO). In the present study, the solvent effects on the order-disorder transition of sclerox with different degrees of carboxylation (DS) in water-DMSO mixtures were investigated with differential scanning calorimetry and optical rotation. The transition temperature ( Tr) and transition enthalpy (Δ Hr) strongly depended on the mole fraction of DMSO ( xD). Data were further analyzed with the statistical theory for the linear cooperative transition, taking into account the solvent effect, where DMSO molecules are selectively associated with the unmodified side chains. The modified side chain does not contribute to the transition; hence, Δ Hr decreases with increasing DS. The dependence of Tr on the DMSO content becomes weaker than that for unmodified schizophyllan. The theoretical analyses indicated that the number of sites binding with the DMSO molecule and the successive ordered sequence of the ordered unit of the triple helix are changed by carboxylation.

Structural characterization of bioactive heteropolysaccharides from the medicinal fungus Inonotus obliquus (Chaga).

The aim of this paper was to perform a comprehensive characterization of polysaccharides isolated from the interior (IOI) and exterior (IOE) parts of the fungus Inonotus obliquus. Pre-extraction with DCM and MeOH, followed by water and alkali extraction and ethanol precipitation gave two water extracts and two alkali extracts. Neutral and acidic polysaccharide fractions were obtained after anion-exchange chromatography of the water extracts. The neutral polysaccharides (60-73 kDa) were heterogeneous and branched and consisted of a (1 → 3)-linked ß-Glc backbone with (1 → 6)-linked kinks in the chain at approximately every fifth residue, with branches of (1 → 6)-linked ß-Glc in addition to substantial amounts of (1 → 6)-linked α-Gal with 3-O-methylation at about every third Gal residue. The acidic polysaccharide fractions (10-31 kDa) showed similar structural motifs as the neutral fractions differing mainly by the presence of (1 → 4)-linked α-GalA and α-GlcA. ß-Xyl, α-Man and α-Rha were also present in varying amounts in all fractions. No major structural differences between the IOI and IOE fractions were observed. An alkaline polysaccharide fraction (>450 kDa) was obtained from the IOI alkali extract, and consisted mainly of (1 → 3)- and (1 → 6)-linked ß-Glc and (1 → 4)-linked ß-Xyl. Several of the fractions showed in vitro immunomodulatory effect by increasing NO production in the murine macrophage and dendritic cell lines J774.A1 and D2SC/1. Most fractions managed to increase NO production only at the highest concentration tested (100 µg/ml), while the neutral fraction IOE-WN activated potent NO production at 10 µg/ml and was considered the most promising immunomodulating fraction in this study.

Macromolecular acidic coating increases shelf life by inhibition of bacterial growth.

The sensitivity of microorganisms to low pH can be utilized in food protection by preparing coatings based on macromolecular acids. Due to limited diffusivity of macromolecules low pH occurs primarily at the surface, while the interior parts of the food remain unaffected. This principle is demonstrated using food approved alginic acid in various types of coatings (aqueous, emulsions, dispersions, dry coating) on a wide range of foods including meat, fish, chicken, shrimp and boiled rice. Significant delay or inhibition of the natural flora is generally demonstrated, particularly when exposed to 'temperature abuse'. Specifically, we show that the coatings reduce or inhibit regrowth of pathogens (Bacillus cereus, B. weihenstephanensis, Listeria monocytogenes serotype 1 and Staphylococcus aureus). In special cases like boiled rice, alginic acid may largely replace acetic acid for acidification and preservation, as demonstrated studying regrowth of added spores of B. cereus. Most formulations allow easy removal prior to further processing (cooking, frying). Temporary side effects such as 'acid cooking' obtained for high acid concentrations on sensitive surfaces (e.g. salmon) disappear during processing, recovering the normal taste and texture. The coating is hence suitable for a large variety of foods.

Relationship between energetic stress and pro-apoptotic/cytoprotective kinase mechanisms in intestinal preservation.

BACKGROUND: A recent study from our laboratory documented significant improvements in post-transplant viability in an experimental model of intestinal transplantation when a novel, nutrient-rich preservation solution was used during cold storage. The current study investigated the relationship between energetic/oxidative stress responses and fundamental kinase signaling events during the period of organ storage. This relationship may be a key factor contributing to improved graft viability after storage in a nutrient-rich preservation solution. METHODS: Rat small intestine was harvested and flushed intraluminally with University of Wisconsin (UW) solution or an amino acid-rich (AA) solution as follows: Group 1, no luminal flush (clinical control); Group 2, luminal UW solution; Group 3, luminal AA solution. Energetics (ATP, total adenylates), oxidative stress (malondialdehyde), histology, and MAPK (P38, JNK, ERK)/AMPK/Caspase-3 were assessed throughout 12-hour cold storage. RESULTS: P38 and JNK were upregulated strongly in Group 2 after 1- and 12-hour storage. Group 3 exhibited a delayed activation and subsequent downregulation of these pre-apoptotic signals. Between 6 to 12 hours, a strong upregulation of ERK was observed in Group 3. AMPK downregulation correlated with a reduction in AMP/ATP ratio, ERK upregulation, and P38/JNK downregulation in Group 3. After 12-hour storage, histology indicated superior preservation of mucosal architecture in Group 3 tissues. CONCLUSIONS: A nutrient-rich preservation solution abrogates pre-apoptotic signaling (JNK and P38) and upregulates cytoprotective signals (ERK). Our data support the concept of a concerted effort facilitating cellular protection in response to ischemic stress.

Effects of carboxylation of the side chains on the order-disorder transition in aqueous solution of schizophyllan, a triple helical polysaccharide.

Schizophyllan and scleroglucan are water-soluble polysaccharides having repeating units consisting of three ß-1,3-linked glucose residues in the main chain and a single ß-1,6-linked glucose residue as the side chain. This polysaccharide dissolves as a triple helix in an aqueous solution and shows a cooperative order-disorder transition between the side chain and solvent molecules while retaining the triple helical conformation. Periodate and subsequent chlorite oxidations selectively modify the side chain glucose to provide the corresponding dicarboxylate units. Optical rotation measurements and differential scanning calorimetry were performed on carboxylated schizophyllan/scleroglucan ('sclerox') samples to investigate the effects of the degree of carboxylation on the order-disorder transition in deuterium oxide with 0.1M NaCl. The transition curves for the sclerox samples are strongly dependent on the degree of carboxylation. The modified side chains cannot take the ordered structure, resulting in a reduction of the transition enthalpy. The transition temperature for carboxylated schizophyllan becomes lowered and the transition curve broadens with increasing the degree of carboxylation. The permanent disordered units are included in a trimer by the carboxylation to inhibit a long sequence of the ordered units.