Hirotaka Kakita

Effects of environmental factors and plant growth regulators on growth of the red alga Gracilaria vermiculophylla from Shikoku Island, Japan

Growth and tolerance of Gracilaria vermiculophylla (Ohmi) Papenfuss from Shikoku Island were investigated under a variation of temperature (5–30 °C), salinity (5–60‰), and photon irradiance (20–100 μmol photons m−2 s−1) in unialgal culture. G. vermiculophylla showed wide tolerances for all factors tested, characterizing a euryhaline and eurythermal species. Two clones, one of a tetrasporophyte and the other of a female gametophyte, showed different growth rates, attributable to the difference either in phase or in genotype. The optimum temperature for the growth of the tetrasporophyte clone was 15–25 °C while that of the gametophyte clone was 20–30 °C. Maximum growth of both phases was observed at 80–100 μmol m−2 s−1. G. vermiculophylla presented higher growth rates in low salinities (15–30‰). Tissue cultures were established in solid ASP 12-NTA medi um supplemented with plant growth regulators (PGR), 0.5% agar, 1.0% sucrose and 0.5% inositol. Effects of two auxins (indole-3-acetic acid (IAA), and 2,4-dichlorophenoxyacetic acid (2,4-D)), and one cytokinin (6-benzylaminopurine (BA)) were tested in concentrations ranging from 0.1 to 10.0 mg l−1. Growth of apical segments was significantly stimulated by the majority of treatments supplemented with PGR, while maximum growth of calluses was observed in treatments with low concentration of auxins or BA (1.0 mg l−1). All treatments supplemented with PGR significantly promoted the growth of intercalary segments, except for IAA (1.0 mg l−1) in combination with BA (1.0 mg l−1). Growth of calluses originating from intercalary segments was observed in treatments with IAA (0.1 mg l−1), 2,4-D (10.0 mg l−1) or IAA (1.0 mg l−1) in combination with BA (0.1 mg l−1). Tr eatments with high concentration of IAA and BA (10.0 mg l−1) were lethal for apical and intercalary segments. These results show that auxin and cytokinin play a regulatory role on the growth of G. vermiculophylla in tissue culture. Furthermore, results on the effects of temperature, salinity and irradiance indicate that G. vermiculophylla could be cultivated in brackish temperate environments with potential for economic purposes and for pollution management.

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.

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.

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.

High-performance affinity chromatography of a chick lectin on an adsorbent based on hydrophilic polymer gel

A mouse monoclonal antibody (SIA4-5) which reacts with a chick 14K lectin (C14K) was covalently attached to a new support for high-performance affinity chromatography, TSKgel Tresyl-5PW, which is a preactivated, polymer-based particle. The immobilized antibody (SIA4-5-5PW) thus prepared proved to be useful in measuring not only the molecular properties of C14K but also specific interactions of C14K with SIA4-5 and hapten sugars. The C14K preparation was fractionated according to the oligomeric structure and with slight differences in affinity to SIA4-5 although the former was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Application of the method for quantitative analytical purposes was successful.

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.