Experimental and theoretical estimation of the Hansen solubility parameters of paramylon esters based on the degrees of substitution and chain lengths of their acyl groups.

Paramylon is a natural hydrophilic polysaccharide produced in the pyrenoids of euglenoids, and esterification may render paramylon hydrophobic. Esterification imparts not only thermoplasticity, but also potential compatibilities with other polymer resins and fillers. However, the dependence of the compatibility on the structure of the polymer ester has not yet been systematically studied. To estimate the affinities between paramylon esters and hydrophobic organic solvents/resins, the dependences of their Hansen solubility parameters, which are association indices, on the degrees of substitution and chain lengths of the ester groups were investigated. Experimental and theoretical investigations were conducted using the dissolution and Fedors methods, respectively. Esterification decreased the solubility parameter from 49 (paramylon) to approximately 18 MPa1/2 (paramylon esters), indicating that the potential affinities of paramylon esters for hydrophobic organic solvents/polymers increased. A multiple regression analysis was also performed to investigate the effects of acyl chain length and degree of substitution with acyl groups on the solubility parameter. The solubility parameters of the paramylon derivatives were continuously variable from hydrophilic to -phobic. Hence, esterification with various acyl groups may control the hydrophobicities of paramylon esters, enhancing their miscibilities with various hydrophobic organic solvents and resins.

Correction to: Creation of Straight-Chain Cationic Polysaccharide-Based Bile Salt Sequestrants Made from Euglenoid ß-1,3-Glucan as Potential Antidiabetic Agents.

The corresponding author (Motonari Shibakami) inadvertently failed to include his ORCID ID: http://orcid.org/0000-0003-4484-2982 In the published article.

Creation of Straight-Chain Cationic Polysaccharide-Based Bile Salt Sequestrants Made from Euglenoid ß-1,3-Glucan as Potential Antidiabetic Agents.

PURPOSE: Straight-chain polysaccharides have a greater potential of selectively adsorbing hydrophobic bile salts than resin-based bile salt sequesters because of ionic and hydrophobic interactions; hence, they may possess antidiabetic activity. The feasibility of using cationic polysaccharides made from euglenoid ß-1,3-glucan (referred to as paramylon) as potential antidiabetic agents was examined by using in vitro and animal experiments. METHODS: Cationic straight-chain polysaccharides were synthesized from euglenoid polysaccharide and glycidyltrimethylammonium chloride. The effects of administration of the synthetic polysaccharide on metabolic syndrome-related indicators were examined in high-fat diet-induced obesity mice. The degree of adsorption of bile salts by the polysaccharides was evaluated using spectroscopic analysis. RESULTS: Administration of the cationic paramylon derivatives significantly reduced body and mesenteric fat weight in high-fat diet-induced obesity mice. A noteworthy effect was that glucagon-like peptide-1 (GLP-1) secretion was approximately three times higher in diet-induced obesity mice receiving cationic paramylon derivatives than in those receiving cellulose as a control. CONCLUSIONS: Our results indicate that these cationic paramylon derivatives are potential GLP-1 secretagogues suitable for further study.

Surface plasmon resonance analysis of interactions between diacylglycerol acyltransferase and its interacting molecules.

To measure the interactions of diacylglycerol acyltransferase (DGAT) by surface plasmon resonance (SPR), we immobilized Saccharomyces cerevisiae DGAT2 encoded by DGA1 on a BIACORE sensor chip surface. We used N-terminally truncated Dga1p with a FLAG tag at the C-terminus, which was purified to apparent homogeneity, maintaining significant DGAT activity (Kamisaka et al., Appl. Microbiol. Biotechnol., 88, 105-115 (2010)). Truncated Dga1p with a FLAG tag was immobilized with an anti-FLAG antibody that had been coupled with an L1 chip surface consisting of a carboxymethyl dextran matrix with additional hydrophobic alkane groups. The Dga1p-immobilized chip surface was analyzed for interactions of Dga1p with oleoyl-CoA, its substrate, and anti-Dga1p IgG, its interacting protein, by SPR. The binding of these analytes with the Dga1p-immobilized chip surface was specific, because butyryl-CoA, which cannot be used as a substrate for DGAT, and anti-glyceraldehyde-3-phosphate dehydrogenase IgG, did not induce any signals on SPR. Furthermore, injection of organic compounds such as xanthohumol, a DGAT inhibitor, into the Dga1p-immobilized chip surface induced significant SPR signals, probably due to interaction with DGAT. Another DGAT inhibitor, piperine, did not induce SPR signals on application, but induced them due to piperine on application together with oleoyl-CoA, in which piperine can be incorporated into the micelles of oleoyl-CoA. The results indicate that the Dga1p-immobilized L1 chip surface recognized DGAT inhibitors. Taking all this together, SPR measurement using the Dga1p-immobilized L1 chip surface provided a useful system to elucidate the structure-function relationships of DGAT and screen DGAT inhibitors.

Self-organized honeycomb-patterned microporous polystyrene thin films fabricated by calix[4]arene derivatives.

Calix[4]arene derivatives bearing carboxyl groups at the upper rim and alkyl groups at the lower rim were synthesized. Micrometer-size porous honeycomb-patterned thin films were prepared by evaporating chloroform solution of polystyrene containing the calixarene derivatives under high humidity. These films were coated on gold electrodes of QCM, and the high-frequency changes were observed to detect volatile organic compounds such as dichlorobenzene.

Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the Deltasnf2 disruptant produces a significant increase in its enzyme activity.

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the Deltasnf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the Deltasnf2 disruptant. To analyze the expression of Dga1p in wild type and the Deltasnf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the Deltasnf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the Deltasnf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the Deltasnf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1DeltaN2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1DeltaN2p is activated Dga1p. Dga1DeltaN2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.

Melt spinnabilities of thermoplastic paramylon mixed esters.

The low thermoplasticities of polysaccharide esters make them unsuitable for melt spinning. In this study, we aimed to overcome this problem by mixed esterification of paramylon, a euglenoid ß-1,3-glucan with short- and medium-chain acyl groups, as melt-spinnable materials. Thermal analyses revealed that all the synthesized paramylon mixed esters exhibited glass transition temperatures greater than 100 °C; some of them showed large differences between the melting and 5%-weight-loss temperatures (Td5s) and are extrudable through a spinneret at a temperature ~100 °C below Td5, rendering them potential candidates for the production of melt-spun filaments. Among the various compounds investigated, paramylon acetate propionates, in which the degrees of acetyl- and propionyl-group substitution were 0.5-0.7 and 2.2-2.5, respectively, could be melt-spun to yield mechanically tough crystalline monofilaments. In contrast, the melt spinning of cellulose acetate propionate, analogous to the paramylon acetate propionates in terms of acyl substituents, their substitution degrees, and molecular weights, but differs from it in terms of the glucose linkage mode (i.e., ß-1,3 vs ß-1,4), yielded brittle, charred, and short filaments. Curdlan acetate propionate, another analogue with a degree of polymerization five times larger than that of paramylon mixed esters, was not extrudable due to the lack of thermoplasticity. Therefore, we herein confirmed the superiority of paramylon as a primary raw material for melt-spun filaments.

Structure of synthetic transmembrane lipid membranes at the solid/liquid interface studied by specular X-ray reflectivity.

We fabricated a new class of supported membranes based on monolayers of artificial bola (transmembrane) lipids. The lipids used in this study are symmetric bola lipids with two phosphocholine head groups, which resemble natural archaea lipids. To prevent bending of the hydrocarbon chains, stiff triple bonds are inserted in the middle of the hydrocarbon cores. The formation of homogeneous "monolayers" of transmembrane lipids over macroscopic areas can be monitored with fluorescence microscopy. Structures of such supported monolayers in bulk water were characterized with specular X-ray reflectivity using high energy X-ray radiation, which guarantees a high transmission through bulk water. Here, the vertical structure of single monolayers could be resolved from reconstructed electron density profiles. To verify the structural model suggested by the specular reflectivity, we also performed small- and wide-angle X-ray scattering of transmembrane lipid suspensions. The wide-angle patterns reflect a distorted chain-chain correlation, while the small-angle scattering allowed us to model an electron density profile which is consistent with the profile calculated from specular reflectivity.

Stereoselective glycosylations using benzoylated glucosyl halides with inexpensive promoters.

Reactions of O-benzoylated glucopyranosyl halide (I, Br), isolated or generated in situ from per-benzoylated glucose (8a) and trimethylsilyl halide, with various alcohols were efficiently promoted by zinc halide (Cl, Br) or N-bromosuccinimide with a catalytic ZnI(2) to give the corresponding 1,2-trans-beta-glucosides in good to high yields. When the anomeric halogenation of 8a was carried out in the presence of reactive alcohols, 1,2-cis-alpha-glucosides were selectively formed.

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles.

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.

Thermal, crystalline, and pressure-sensitive adhesive properties of paramylon monoesters derived from an euglenoid polysaccharide.

The thermal, crystalline, and pressure-sensitive adhesive properties of thermoplastic monoesters made from paramylon, a storage polysaccharide of Euglena gracilis, and a long-chain acyl chloride, were examined. Differential scanning calorimetry revealed that the thermal properties of these paramylon monoesters were dependent on the chain length and the average degree of substitution of the long-chain acyl group (av. DSlca). X-ray diffractometry revealed that the product solids with a myristoyl or palmitoyl group had a less ordered lateral acyl chain structure than those with a stearoyl group. Tackiness testing showed that the introduction of a myristoyl group into paramylon with an av. DSlca of ∼2.6 to ∼2.9 yielded palpable pressure-sensitive adhesion. A slight deviation of the chain length and/or av. DSlca from those of tacky paramylon myristate solids weakened or dispersed the tackiness. These results demonstrate the feasibility of using paramylon myristate solids with the av. DSlca in a specific range as a practical pressure-sensitive adhesive.

Construction of higher-ordered monolayer membranes derived from archaeal membrane lipid-inspired cyclic lipids with longer alkyl chains.

A series of artificial cyclic lipids that mimic archaeal membrane ones has been synthesized. The structural features of these molecules include a longer cyclic framework, in which the alkyl chain length ranges from 24 to 32 in carbon number, which is longer than our first analogous molecule with 20-carbon long alkyl chains [K. Miyawaki, T. Takagi, M. Shibakami, Synlett 8 (2002) 1326]. Microscopic observation reveals that these molecules have a self-assembling ability: hydration of the lipids yields multilamellar vesicles in aqueous solution and monolayer sheets on solid supports. High-sensitivity differential scanning calorimetry (24- and 28-carbon alkyl chain lipids) indicates that (i) the alkyl chain length affects their phase behavior and (ii) the enthalpies of endothermic peaks accompanied by phase transition were considerably lower than those of their monomeric phospholipid analogs. Fluorescence polarization measurements suggest that the membranes made from the 24-carbon alkyl chain lipid have a higher polarization factor than membranes composed of DMPC and DMPC plus cholesterol. These findings imply that the cyclic lipids containing 24- and 28-carbon alkyl chain construct well-organized monolayer membranes and, in particular, that the molecular order of the 24-carbon alkyl chain lipid is higher than that of bilayer membranes in the liquid-ordered phase.

Thickening and water-absorbing agent made from euglenoid polysaccharide.

Paramylon, a storage polysaccharide of Euglena gracilis, is a linear ß-1,3-glucan with a weight-average molecular weight of ∼2.0×105. Sequential long-chain acylation and succinylation of paramylon yielded amphiphilic paramylon acylate succinates. Owing to their amphiphilicity, these paramylon derivatives showed higher viscosity than paramylon succinate when dispersed in an aqueous solution. Examination of the viscosity of aqueous solutions containing paramylon acylate succinates differing in chain length and degree of substitution of long-chain acyl groups (DSlca) revealed that the longer the acyl chain and the higher the DSlca, the higher the viscosity of the aqueous solution. Solution casting yielded transparent and mechanically tough films from paramylon acylate succinates. These films had high water absorbability, up to ∼1000 times their weight.

Synthesis and thermal properties of paramylon mixed esters and optical, mechanical, and crystal properties of their hot-pressed films.

Acylation of paramylon, a storage polysaccharide of Euglena gracilis, using multiple acid anhydrides yielded thermoplastic paramylon mixed esters without significant depolymerization. DSC examination showed that the shorter the acyl chain, the higher both the melting and glass transition temperature of the ester. TG analyses revealed their higher thermostability with the 5% weight loss temperature of ∼330°C. Melt volume flow rate examination revealed that the longer the acyl chain, the higher the thermoplasticity of the ester and that the esters exhibited higher thermoplasticity than structurally analogous esters made from cellulose and curdlan. A notable feature of the thermoplastic paramylon mixed esters is the availability of hot-pressing as a means of molding them into a film. Light transmittance and XRD measurements revealed that these films were transparent and in the amorphous state. Tensile tests indicated that the films had adequate mechanical strength comparable to those of the cellulose and curdlan analogues.

Synthesis of nanofiber-formable carboxymethylated Euglena-derived ß-1,3-glucan.

Carboxymethylation of paramylon, a storage polysaccharide of Euglena, was explored using homogeneous and heterogeneous reaction systems. Homogeneous reactions yielded carboxymethyl paramylons with a low degree of substitution of carboxymethyl group (DScm) of ∼0.13. Heterogeneous reactions of paramylon with chloroacetic acid in a mixture of 2-propanol and a NaOH aqueous solution yielded carboxymethyl paramylons with a high DScm of up to ∼0.80. An essential step for successful carboxymethylation using the heterogeneous reaction systems devised in this study was the pretreatment of a paramylon particle to break its inherent highly crystalline state into an amorphous one. A high degree of substitution provided carboxymethyl paramylons with a nanofiber-forming ability in the aqueous phase while a low degree provided them with a fiber-forming ability in both the aqueous and solid phases.

Preparation of transparent self-standing thin films made from acetylated euglenoid ß-1,3-glucans.

The feasibility of using acetylparamylon to prepare self-standing optical films was investigated. Its thermal stability, represented by the glass transition temperature, and 5% weight loss temperature are equivalent to those of acetylcellulose. The mechanical properties (maximum stress, elongation at break, and elastic modulus) of acetylparamylon films prepared by solution casting were comparable to those of acetylcellulose films. The degrees of their transparency and birefringence were the same as those of the acetylcellulose film. These results, together with the finding that acetylparamylon with a higher degree of acetylation formed a thin film with fewer micrometer-sized defects and that acetylparamylon with a lower degree of acetylation formed a thin film with a nanometer-level flat surface, suggest that acetylparamylon is a feasible material for self-standing optical film.

One-pot synthesis of thermoplastic mixed paramylon esters using trifluoroacetic anhydride.

Mixed paramylon esters prepared from paramylon (a storage polysaccharide of Euglena), acetic acid, and a long-chain fatty acid by one-pot synthesis using trifluoroacetic anhydride as a promoter and solvent were shown to have thermoplasticity. Size exclusion chromatography indicated that the mixed paramylon esters had a weight average molecular weight of approximately 4.9-6.7×10(5). Thermal analysis showed that these esters were stable in terms of the glass transition temperature (>90°C) and 5% weight loss temperature (>320°C). The degree of substitution of the long alkyl chain group, a dominant factor determining thermoplasticity, was controlled by tuning the feed molar ratio of acetic acid and long-chain fatty acid to paramylon. These results implied that the one-pot synthesis is useful for preparing structurally-well defined thermoplastic mixed paramylon esters with high molecular weight.

Thermoplasticization of euglenoid ß-1,3-glucans by mixed esterification.

We experimentally demonstrated that paramylon, a storage polysaccharide of Euglena gracilis, is efficiently thermoplasticized by adding acyl groups that differ in alkyl chain length. Glass transition temperature of mixed paramylon esters was higher than those of plant-based polylactic acid (PLA), poly 11-aminoundecanoic acid (PA11), and petroleum-based acrylonitrile-butadiene-styrene (ABS) resin and was comparable to that of cellulose acetate stearate (CAS). Their thermoplasticity was equivalent to or higher than those of these reference plastics. The bending strength and bending elastic modulus of injection molded test specimens made from mixed paramylon esters were comparable to those of the reference plastics. While their impact strength was lower than that of specimens made from ABS resin and CAS, it was comparable to those of PLA and PA11. Euglenoid ß-1,3-glucans are thus a potential component of thermoplastic materials.

Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid ß-1,3-glucans.

This paper introduces a new strategy for creating surface modified polysaccharide nanofibers. To demonstrate proof of principle, the synthesis, structure, and self-assembly behavior of a carboxylic acid-bearing polysaccharide made from paramylon (ß-1,3-glucan) and succinic anhydride were investigated. Examination by a combination of NMR, FT-IR, and SEC-MALLS confirmed that successful preparation of the desired succinylated paramylon without significant depolymerization. NMR, SEC-MALLS, visible absorption and CD spectroscopic analyses indicated that the paramylon derivative forms the triplex structure in solutions. SEM observation revealed that succinylated paramylon forms a nanofiber that has carboxylic acid on the surface.

Polysaccharide nanofiber made from euglenoid alga.

We have fabricated a polysaccharide nanofiber made from paramylon (ß-1,3-glucan), a storage polysaccharide stored as a micrometer-sized particle in the cell of euglenoid alga. Preparation of this nanofiber primarily hinges on the bottom-up approach. First, paramylon, which is originally present in the form of a bundle of nanofibers in a particle, was fibrillated to a randomly coiled polymer by dissolving the particle in a 1.0-mol/L NaOH aqueous solution. Second, the randomly coiled polymer was allowed to self-assemble into a triplex as the NaOH concentration was reduced to 0.25-0.20mol/L. Third, a 20-nm-width nanofiber made from the triplex emerged in the solution when the NaOH concentration was reduced to approximately 0.20mol/L.