Yoshimi Kanzawa

Difference of molecular association in two types of curdlan gel

Abstract The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures. The neutralized and 60°C-set preparations were soluble in 0·01 m sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 m . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.

Bacillus curdlanolyticus sp. nov. and Bacillus kobensis sp. nov., which hydrolyze resistant curdlan.

Taxonomic characteristics of seven bacterial strains which were isolated from soil and hydrolyze resistant curdlan were studied. These bacteria were aerobic, spore-forming rods, contained menaquinone 7 as a major quinone, contained anteiso-C15:0 and iso-C16:0 as major cellular fatty acids, had guanine-plus-cytosine contents of 50 to 52 mol%, and could be divided into two groups on the basis of physiological and chemotaxonomic characteristics and DNA-DNA hybridization data. We propose the following two new species: Bacillus curdlanolyticus for strains YK9, YK121, YK161, YK201, and YK203, with type strain YK9 (= IFO 15724); and Bacillus kobensis for strains YK205 and YK207, with type strain YK205 (= IFO 15729).

Molecular Association and Dissociation In Formation of Curdlan Gels

We examined the formation of curdlan gels by differential scanning calorimetry and electron microscopy. When curdlan heated in water to various temperatures was cooled, an exo-thermic peak appeared at about 40 °C, with formation of hydrogen bonds involving water to give gel. When the cooled preparations were heated again, endo-thermic peaks reappeared at about 60°C and exo-thermic peaks also appeared at higher temperatures. However, no exo-thermic peaks were observed on cooling and then heating above 145 °C. Thus, the gel formation caused by heating to 60-120 °C and cooling is reversible, but that caused by heating to above 145 °C and cooling is irreversible as shown by the heat analysis. In neutralized gel and the gel formed by heating at 55 or 60 °C and cooling, microfibrils of endless length consisting of fibrils of about 100 nm length and 10-25 nm width appeared. In gels obtained by heating at 70, 80, and 100 °C and cooling, microfibrils with released fine stubs appeared. Gels cooled after heating at 120 and 145 °C contained spindle shaped microfibrils of about 100 nm length and 30 nm width. Preparations obtained by heating to 170 °C and cooling gave microfibrils like those formed by unheated preparations.

Purification and properties of a new exo-(1→3)-β-D-glucanase from Bacillus circulans YK9 capable of hydrolysing resistant curdlan with formation of only laminari-biose

A (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.6), capable of hydrolysing resistant curdlan, was purified chromatographically from the culture supernatant of Bacillus circulans complex YK9 on Toyopearl HW-55F and butyl-Toyopearl 650M columns. The purified enzyme had a specific activity of 190 units mg-1 on regenerated curdlan. The molecular mass was estimated to be about 70 kDa as judged by SDS-PAGE. The enzyme had a pH optimum of approximately pH 6.0. It hydrolysed regenerated and resistant curdlans yielding predominantly laminari-biose, although the rate of hydrolysis of the former was much higher than the latter. This enzyme rapidly hydrolysed laminaran, curdlan and carboxymethyl-curdlan, but did not cleave schizophyllan and screloglucan, which have glucosyl side chains. The enzyme hydrolysed low molecular mass (1-->3)-beta-D-glucans-(mean degree of polymerization, DPn = 131, 49 and 14) and laminari-heptaose more efficiently than curdlan. It also hydrolysed laminari-hexaose and -pentaose effectively, but laminari-tetraose only slightly and it did not hydrolyse laminari-triose or -biose. The enzyme is an exo-hydrolase of curdlan and various oligomers composed of (1-->3)-beta-D-glucosidic linkages, liberating laminari-biose from their non-reducing terminals. The laminari-biose generated was in the alpha-form.