Shiho Suzuki

Structure, physical, and digestive properties of starch from wx ae double-mutant rice.

Amylopectin is the principal component of starch. The amylose extender (ae) gene encodes the starch-branching enzyme IIb, which is critical in determining the fine structure of endosperm starch. To determine the relationship between the fine structure of amylopectin and its physical properties, rice mutant lines defective in the ae function with altered fine structure of amylopectin and in combination with the waxy (wx) background were selected for comparative studies with primary wild-type and ae starches. The ae mutant endosperms accumulated a high amylose content starch with long amylopectin chains. The ae and wx ae starches showed no significant difference in the unit chain-length distribution of amylopectin and starch granule morphology. The wx ae starch displayed a higher pasting temperature and higher peak viscosity. The gelatinization peak temperatures of the wx, ae, and wx ae starches were 2.2, 13.1, and 17.1 degrees C higher, respectively, than that of the wild-type starch, and the wx ae starch showed a retrogradation peak with a shorter cooling period than that of ae starch. The raw ae and wx ae starches were almost indigestible by alpha-amylase in vitro. Rats fed the wx ae starch showed slowly increasing blood glucose at a lower level than the rats fed the wx or wild-type starch. These results indicate that the primary structure of the rice wx ae amylopectin with enriched long chains changes the granular structure of the starch, including its crystal structure, and results in resistance to in vitro or in vivo degradation.

Dependence of the mechanical properties of a pullulan film on the preparation temperature

The mechanical properties of pullulan films prepared at various temperatures were investigated. The films prepared at high temperatures (40°C and 60°C; H-films) did not show any clear plastic deformation in tensile test, indicating that they were brittle. In contrast, those prepared at low temperatures (4°C, 13°C, and 25°C; L-films) showed such deformation. The latter films had higher values for both tensile strength and elastic modulus than the former, indicating that the L-films were stiffer and more flexible than the H-films. Stretching the L-films clearly showed a shear deformation band inclined at 45° to the stretching direction, indicating that they were amorphous.

Primary structure, conformation in aqueous solution, and intestinal immunomodulating activity of fucoidan from two brown seaweed species Sargassum crassifolium and Padina australis.

We studied the structure of fucoidans extracted from two brown seaweed species, Sargassum crassifolium and Padina australis, and their intestinal immunomodulating activity via Peyers patch cells of C3H/HeJ mice. ESI-MS analysis indicated that the dominant structure of both fucoidans has a backbone of α-(1→4)-linked and α-(1→3)-linked l-fucose residues and sulfate groups are attached at the C-2 and C-4 positions; branches of fucoidan from S. crassifolium are galactose residues with (1→4)- linkage and branching points are at C-4 of fucose, while fucoidan from P. australis, branches are sulfated galactose-fucose disaccharides and sulfated galactose monosaccharides attached to the main chain through (1→3)- or (1→4)- linkages. According to small angle X-ray scattering (SAXS) measurements, the two fucoidans have a branched structure. We simulated them with molecular models based on our proposed primary structure. These fucoidan samples have the ability to stimulate intestinal immunological activity via Peyers patch cells.

Fourth 3D Structure of the Chitosan Molecule: Conformation of Chitosan in Its Salts with Medical Organic Acids Having a Phenyl Group

Chitosan salts with two medical organic acids having phenyl groups (salicylic and gentisic acids) exhibited fiber diffraction patterns of a new type of crystal which does not compare with known types I and II. The crystals, called type III salts, showed a fiber repeat of 2.550 nm and a meridional reflection at the 5th layer line. These results coupled with a conformational analysis indicate the chain conformation of chitosan with the salts to be a 5/3 helix, this helix differing from those of type I (an extended two-fold helix) and type II (a relaxed two-fold helix or a 4/1 helix). The fiber patterns of all the type III salts were similar. This observation has also been found with type II salts and is an indication that the acid ions are not arranged in regular positions in the crystals. A comparison of solid-state 13C-NMR spectra of the gentisic acid salt and the aspirin salt, which could not be crystallized, suggests that, in the latter salt, the chitosan molecules also formed a 5/3 helix.

Characterization of Fusarium oxysporum β-1,6-Galactanase, an Enzyme That Hydrolyzes Larch Wood Arabinogalactan

ABSTRACT A type II arabinogalactan-degrading enzyme (FoGal1) was purified from Fusarium oxysporum 12S, and the corresponding cDNA was isolated. FoGal1 had high similarity to enzymes of glycoside hydrolase family 5. Treatment of larch wood arabinogalactan with the recombinant enzyme indicated that FoGal1 is a β-1,6-galactanase that preferentially debranches β-1,6-galactobiose from the substrate.

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Background: Cycloisomaltooligosaccharide glucanotransferase catalyzes an intramolecular transglucosylation reaction and produces cycloisomaltooligosaccharides from dextran. Results: The crystal structure of Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase was determined. Conclusion: The enzyme structures complexed with isomaltooligosaccharides and cycloisomaltooctaose revealed the molecular mechanism of action. Significance: CBM35 functions in the product size determination and substrate recruitment. Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)8-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite −8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.

Conformation and physical properties of cycloisomaltooligosaccharides in aqueous solution.

We studied the conformation and physical properties of cyclic and linear isomaltooligosaccharides in aqueous solution by intrinsic viscosity measurement, small angle X-ray scattering (SAXS) and molecular modeling. We used four cycloisomaltooligosaccharide samples (CIs) with degree of polymerization (DP) 7-10 (CI-7-CI-10) and five linear isomaltooligosaccharide samples (LIs) with DP 7-11 (LI-7-LI-11). The values of α in the Mark-Houwink-Sakurada equation [η]=KM(w)(α) for the CI and LI were determined to be 0.50 and 0.78, respectively. The radii of gyration (R(G)) of CI-7, CI-8, CI-9 and CI-10 determined from SAXS data were 6.7, 6.9, 7.5 and 8.3Å, respectively. The scattering profile of CI-9 compared with those obtained for molecular models indicated that CI molecular chains are less flexible than those for LIs and adopt a rather compact circular conformation.

Nanostructure and physical properties of cellulose nanofiber-carbon nanotube composite films

We studied the nanostructure and physical properties of cellulose nanofiber-multi-walled carbon nanotube (CNF-MWNT) composite films prepared via MWNT aqueous dispersion using 4-O-methyl-α-d-glucuronoxylan as a MWNT dispersion aid. The composite film had high electrical conductivity (1.05S/cm), good mechanical properties (Youngs modulus: 10.1GPa, tensile strength: 173.4MPa) and a low coefficient of thermal expansion (7ppm/K). FE-SEM imaging showed that the carbon nanotubes dispersed homogeneously and made reinforcing networks in the matrix of cellulose nanofibers. Improvement in the physical properties of cellulose nanofiber film by adding MWNTs is due to this composite structure.

Gamma-Crosslinked Collagen Gel without Fibrils : Analysis of Structure and Heat Stability

This paper reports an analysis of the structure and heat stability of two different collagen gels: conventional collagen gel (neutral gel) and gel without collagen fibrils (acidic gel), previously reported. We performed differential scanning calorimetry (DSC), observations by scanning electron microscope (SEM), observations by atomic force microscope (AFM), and small angle X-ray scattering (SAXS). Collagen fibrils were clearly observed in the neutral gel but not in the acidic gel by both SEM and AFM. A clear endothermic peak was observed at 53–55 °C, representing disassembly of collagens in collagen fibrils in the neutral gel but not in the acidic gel. Only a small broad endothermic peak, at 35–43 °C, representing the deformation of the triple helical structure of collagen, was observed in the acidic gel. The SAXS pattern also suggested that the neutral gel had a more heterogeneous structure than the acidic gel. The experimental results described here are compatible with the model proposed in a previous paper, and indicate more clearly that the acidic gel has no collagen fibrils and has a different molecular assembly state of Type I collagen than the neutral gel.

A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain

We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with α-d-galactopyranosidase (GPase) and β-l-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from α-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with α-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of α-galactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and α-l-arabinofuranosidase released four times more arabinose than α-l-arabinofuranosidase acting alone. This suggested that a large number of α-l-arabinofuranosyl residues is capped by α-galactosyl residues. 1H NMR experiments revealed that rFoGP1 hydrolyzed the α-1,3-galactosidic linkage within the side chain structure of [α-d-Galp-(1→3)-α-l-Araf-(1→] in gum arabic. In conclusion, rFoGP1 is highly active toward α-1,3-galactosyl linkages but negligibly or not active toward α-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.