Hiroshi Kamishima

Simultaneous analysis of monosaccharides and oligosaccharides by high-performance liquid chromatography with postcolumn fluorescence derivatization.

To develop a fluorimetric HPLC technique for the simultaneous microanalysis of reducing mono- and oligosaccharides, the technique of linear gradient elution was introduced into the postcolumn fluorimetric detemination system of reducing saccharides with benzamidine. Fluorescence measurement was performed at 288 nm for excitation and 470 nm for emission and an optimization study for this postcolumn fluorescence derivatization carried out. Under optimum conditions, the detection limits of D-glucose and maltohexaose were 1.78 and 2.59 pmol, respectively. The present method was successfully applied to saccharide analysis and should prove useful for automated simultaneous microanalysis of reducing mono- and oligosaccharides in foods.

Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a β-1′, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3′ and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3′ differentiates E. carotovora lipid A from that of other gram-negative bacteria.

Effects of Environmental Factors and Metal Ions on Growth of the Red Alga Gracilaria Chorda Holmes (Gracilariales, Rhodophyta)

Gracilaria is a potentially valuable source of marine biopolymers such as proteins and polysaccharides. In order to select suitable culture conditions, growth and tolerance of Gracilaria chorda Holmes from Shikoku Island in southwest Japan were investigated under variations of temperature (5–30 ∘C), photon irradiance (20–120 μmol photons m−2 s−1), and photoperiod (12:12 h, 14:10 h light:dark regime) in a unialgal culture. Gracilaria chorda showed wide tolerances for all factors investigated, which is characteristic of eurythermal species. Maximum growth was observed at 18–24 ∘C. The optimum photon irradiance for the algal growth was 60–120 μmol photons m−2s−1. Instead of using ordinary sea salt (NaCl) to prepare artificial seawater, ultra pure salt was adopted. Gracilaria chorda grew faster in artificial seawater made with ultra-pure salt than that made with ordinary sea salt, probably because the former medium was clear, while the latter was milky. Effects of some metal ions on the growth were tested with artificial seawater. Iron ions affected algal growth, but cobalt ions did not. This study enables us to determine suitable culture conditions for G. chorda. A scaled-up 30 l culture of G. chorda under such conditions was successful.

Extracellular lipopolysaccharide production by Erwinia carotovora

Abstract The plant-pathogenic bacterium Erwinia carotovora FERM P-7576 produced lipopolysaccharide (LPS) in culture broth. A combination of two carbon sources, such as pectin and other carbohydrates, promoted LPS production. The organism produced the LPS during exponential growth. Production of the LPS was influenced by the cultivation method and temperature. About 920 mg per l of LPS was obtained by two-stage cultivation, in which the bacterium was grown on pectin medium at the end of the exponential growth phase, then glycerol and sodium l -glutamate were added to continue the cultivation. The maximum productivity was obtained at 33°C. The extracellular LPS was essentially identical to the cellular LPS isolated from the cells, on the basis of chemical compositions, as analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, and endotoxic activity.

Purification and Properties of a High Molecular Weight Hemagglutinin from the Red Alga, Gracilaria verrucosa

A high molecular weight hemagglutinin was purified from a phosphate buffer extract of the red alga, Gracilaria verrucosa, by ammonium sulfate precipitation, followed by ion exchange and gel filtration chromatographies. Its molecular weight was estimated by gel filtration to be approximately Afr480000. It contained large amounts of hexose and sulfate along with a small amount of protein. Because the acidic polysaccharide demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) had hemagglutinating activity, the agglutinin was assumed to be sulfated polysaccharide with low protein content. Its erythrocyte specificity is different from those of other G. verrucosa hemagglutinins as it agglutinated rabbit and guinea pig erythrocytes, but was inactive against horse erythrocytes. Similar to many other marine algal hemagglutinins, it agglutinated a sheep erythrocyte suspension treated with pronase (SETP), but had no affinity for monosaccharides, and had no divalent cation requirement for hemagglutination. However, it was different from other algal hemagglutinins with regards to heat-durability and periodate-sensitivity. These results suggest that the hemagglutinin is a new component found in the buffer extract of G. verrucosa.

Isolation and characterisation of a fourth hemagglutinin from the red alga, Gracilaria verrucosa , from Japan

Isolation and characterisation of marine algal hemagglutinins or lectins are essential for their potential industrial application as specific carbohydrate affinity ligands. The phosphate buffer extract of the red alga, Gracilaria verrucosa (Huds.) Papenfuss (Gigartinales, Rhodophyta) from Japan is known to contain three different hemagglutinins. The extract of the alga collected in March 1993 from Kagawa Prefecture, Japan, was purified by ammonium sulphate fractionation, ion exchange and gel filtration chromatography. Using gel filtration, two peaks were obtained (hereafter Peak 1 and Peak 2) which differed in molecular size and hemagglutinating activity against horse erythrocytes. Peak 1 corresponded to the known high molecular weight hemagglutinin, H-GVH. Peak 2 contained large amounts of hexose and sulphate along with a small amount of protein. It had a low molecular weight (gel filtration) similar to that of two of the previously reported G.verrucosa hemagglutinins but differed in its electrophoretic behaviour. Peak 2 is therefore a fourth hemagglutinin. Its activity was not inhibited by any of the monosaccharides tested but by the complex glycoproteins such as asialofetuin and fetuin. It had no divalent cation requirement for hemagglutination. The properties of this novel hemagglutinin could prove useful in industrial applications.

Facile isolation of endo-pectate lyase from Erwinia carotovora based on electrostatic interaction.

Endo-pectate lyase (PATE) fromErwinia carotovora was selectively cosedimented with extracellularly produced lipopolysaccharide-lipid complex (LPSLC) through dialysis of the cell free culture broth. The selective isolation of PATE was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cosedimentation of the PATE with LPSLC was initiated by decreasing conductivity of the solution and terminated at approx 1 m Siemens (mScm-1). As much as 62% of PATE activity in the culture broth was removed by precipitation. PATE was isolated from the precipitate by gel chromatography. The cosedimentation of PATE with LPSLC was remarkably affected by pH or ionic strength. The addition of polycationic peptide polymyxin B sulfate or a metal chloride affected the interaction. The cosedimentation was diminished by acetylation of the free amino groups of PATE. From these results, it was confirmed that the cosedimentation was induced by electrostatic interaction.

Inhibitory effect of red alga lectin and skipjack fat on the growth of the red tide plankton Chattonella antiqua

Abstract Lectin prepared from Gracilaria verrucosa and fish fat extracted from skipjack muscle were investigated for their activities against the growth of the red tide plankton Chattonella antiqua . Addition of the seaweed lectin at 50 μg/ml altered the morphology of the plankton cells, which became shrunken form, and their growth was completely suppressed. Skipjack fat not only inhibited microalgal growth but also destroyed the cells within 5 min at 20 μg/ml. These marine materials are promising candidates for restraining the action of red tide planktons.

Purification and some properties of a d-fructose-1,6-bisphosphate aldolase from the red alga, Gracilaria chorda Holmes

Abstract D-Fructose-1,6-bisphosphate aldolase (0.042 mg) was purified from a red alga, Gracilaria chorda, by ammonium sulfate precipitation, followed by ion exchange and hydrophobic interaction chromatographies. The enzyme was purified 87-fold resulting in a final specific activity of 10.7 units/mg and a homogeneous appearance on electrophoresis. By SDS-PAGE analysis of the enzyme, two protein bands corresponding to molecular masses of 45 kDa and 39 kDa were observed. The enzymic activity disappeared in the presence of 0.5 mM EDTA, but was restored to 109% of the original untreated activity by the addition of 1 mM ZnSO4 subsequent to EDTA treatment. The enzyme was activated up to 5.5 times the original untreated activity by the addition of 500 mM KCl, and had a narrow optimum pH around 7.2. On the basis of these results, the G. chorda D-fructose-1,6-bisphosphate aldolase was characterized as a class II aldolase.