Synthesis and properties of fatty acid starch esters

Being completely bio-based, fatty acid starch esters (FASEs) are attractive materials that represent an alternative to crude oil-based plastics. In this study, two synthesis methods were compared in terms of their efficiency, toxicity and, especially, product solubility with starch laurate (C₁₂) as model compound. Laurates (DS > 2) were obtained through transesterification of fatty acid vinylesters in DMSO or reaction with fatty acid chlorides in pyridine. The latter lead to higher DS-values in a shorter reaction time. But due to the much better solubility of the products compared to lauroyl chloride esterified ones, vinylester-transesterification was preferred to optimize reaction parameters, where reaction time could be shortened to 2 h. FASEs C₆–C₁₈ were also successfully prepared via transesterification. To determine the DS of the resulting starch laurates, the efficient ATR-IR method was compared with common methods (elementary analysis, ¹H NMR). Molar masses (M_w) of the highly soluble starch laurates were analyzed using SEC-MALLS (THF). High recovery rates (>80%) attest to the outstanding solubility of products obtained through transesterification, caused by a slight disintegration during synthesis. Particle size distributions (DLS) demonstrated stable dissolutions in CHCl₃ of vinyl laurate esterified – contrary to lauroyl chloride esterified starch. For all highly soluble FASEs (C₆–C₁₈), formation of concentrated solutions (10 wt%) is feasible.     

Assembly of a 20-nm Protein Cage by Escherichia coli 2-Hydroxypentadienoic Acid Hydratase

The pentameric Escherichia coli enzyme 2-hydroxypentadienoic acid hydratase assembles to form a 20-nm-diameter particle comprising 60 protein subunits, arranged with 532 symmetry when crystallised at low pH in the presence of phosphate or sulphate ions. The particles form rapidly and are stable in solution during gel filtration at low pH. They are probably formed through trimers of pentamers, which are stabilised by the interaction of two phosphate ions with residues of the N-terminal domains of subunits at the 3-fold axis. Once the particles are formed at high concentrations of phosphate (or sulphate), they remain stable in solution at 20-fold lower concentrations of the anion. Guest molecules can be trapped within the hollow protein shell during assembly. The C-termini of the subunits are freely accessible on the surface of the protein cage and thus are ideal sites for addition of affinity tags or other modifications. These particles offer a convenient model system for studying the assembly of large symmetrical structures and a novel protein nanoparticle for encapsulation and cargo delivery.     

Acetylation and characterization of spruce (Picea abies) galactoglucomannans

Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, ¹H and ¹³C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA.     

Preparation and characterisation of chitosans with oligosaccharide branches

The trimer 2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1-->4)-2-acetamido-2-deoxy-D-glucopyranosyl-beta-(1-->4)-2,5-anhydro-D-mannofuranose (A-A-M) was reductively N-alkylated onto a fully de-N-acetylated chitosan (F(A)<0.001, DP(n)=25) to obtain branched chitosans with degree of substitution (DS) of 0.070, 0.23 and 0.40, as determined by 1H NMR spectroscopy. The apparent pK(a) values of the primary and secondary amines of the chitosans substituted with the trimer A-A-M were determined by monitoring the chemical shift of the H-2 of GlcN, and were determined as 6.5-6.9 for the primary (unsubstituted) amines and as 5.0-5.2 for the secondary (substituted) amines. The intrinsic pK(a) values (pK(int)) were found to be 7.3-7.4 for the substituted and 8.7 for the unsubstituted amines. The chitosan branched with A-A-M (DS 0.40) was found to be soluble in aqueous solution over the entire pH range. SEC-MALLS (size-exclusion chromatography with a multi-angle laser light scattering detector) further showed that addition of branches did not affect the molar hydrodynamic volume of the chitosan.     

Periodate oxidation of chitosans with different chemical compositions

Periodate oxidation of chitosans with different chemical compositions were investigated by determining the consumption of periodate consumed, and the amount of ammonia and formaldehyde liberated during the reaction. Oxidised chitosans were further characterised by size-exclusion chromatography with online multi-angle light scattering (SEC-MALLS) to obtain the molecular weight distributions, and by elemental analysis to obtain the N/C ratio. Chitosans became only partially oxidised by periodate, reaching degrees of oxidation around 0.5, when oxidising with excess periodate. Overconsumption of periodate is attributed to the extensive depolymerisation, which occurs concomitantly with the oxidation, thereby exposing novel reducing and non-reducing ends which consume additional periodate. Both the rate and extent of overoxidation, and the rate of depolymerisation decreased with increasing F(A). A chitosan-specific degradation mechanism is probably involved in the depolymerisation in addition to the general free-radical-mediated degradation.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

Effect of microwave irradiation on the molecular and structural properties of hyaluronan

Hyaluronan (Na⁺ salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500 min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (M_w) of HA decreased from 1.44 × 10⁶ to 5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the M_w and intrinsic viscosity data according to the Mark–Houwink equation, [η] = k × M_w^α suggested three relationships with α₁ = 0.46 for M_w > 500,000, α₂ = 0.84 for M_w between 500,000 and 50,000, and α₃ = 1.13 for M_w < 50,000. The results revealed that HA with M_w > 10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105 °C. At reaction times larger than 240 min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.     

Probing macromolecular architectures of nanosized cyclic structures of (1-->3)-beta-D-glucans by AFM and SEC-MALLS

Comb-like branched (1-->3)-beta-D-glucans dissolve in water as stiff triple-helical structures. Dissociation followed by re-association leads to the formation of a blend of various macromolecular topologies, where the cyclic species make up a significant fraction. In this study, the molecular properties of these nanosized cyclic structures of (1-->3)-beta-D-glucans were probed using a combination of AFM and SEC-MALLS. The cyclic structures were obtained by subjecting linear triple-helical molecules of (1-->3)-beta-D-glucans to a denaturation-renaturation cycle, and the fraction of cyclic structures in the renatured sample was determined by AFM. Samples containing different known fractions of linear and circular molecules were studied by SEC with online multi-angle laser-light scattering and viscometric detectors. The molecular weight and the radius of gyration of the molecules eluting from the SEC column, as well as the concentration and the intrinsic viscosity, were determined simultaneously. By extrapolating the results to a situation of only circular species, the results allowed to determine the linear mass per unit length (M(L)) of not only the linear but also the circular morphologies of the (1-->3)-beta-D-glucans. The values obtained were M(L)=2140+/-180 g mol(-1)nm(-1) for the circular species and 2045+/-80 g mol(-1)nm(-1) for the linear species. This is the first direct determination of the M(L) parameter of the circular topology, and the results indicate that the reassociation of the individual chains yield a triplex structure also for the circular morphology, similar to the initial triple helix.     

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.