Elsevier

Carbohydrate Polymers

Volume 80, Issue 2, 12 April 2010, Pages 491-497
Carbohydrate Polymers

Crystal transition of paramylon with dehydration and hydration

https://doi.org/10.1016/j.carbpol.2009.12.009Get rights and content

Abstract

The structure of paramylon, a highly crystalline (1  3)-β-d-glucan material, was analyzed by synchrotron X-ray powder diffraction and solid-state 13C NMR spectroscopy. Native paramylon, the hydrate with high crystallinity, was converted into the anhydrous form by drying, by which the crystallinity decreased and the molecular conformation became partially disordered. The anhydrous readily resumed the hydrate form by immersing in water, but the crystal size and the homogeneity of molecular conformation did not reach those of the native hydrate. Crystal transition between the hydrate and anhydrous by relative humidity (R.H.) changes was monitored by X-ray diffractometry and gravimetry. The dehydration occurred around R.H. 30%, and the re-hydration occurred around R.H. 70%. The changes in weight and unit-cell volume by dehydration allowed determination of the number of water molecules in the hydrate as one per anhydroglucopyranoside residue.

Introduction

(1  3)-β-d-Glucan is a polysaccharide occurring in many organisms such as fungi, bacteria, algae and annual plants (Stone & Clarke, 1992). It has attracted much attention in the medical field due to its physiological effects such as immunomodulation and anti-cancer activities (Ooi & Liu, 2000). While many (1  3)-β-d-glucans have branches by β-(1  6) linkages, two organisms are known to produce strictly linear (1  3)-β-d-glucan; those are curdlan, the extracellular polysaccharide of Alcaligenes bacteria, and paramylon, the disk-like storage granule in Euglena cells (Clarke and Stone, 1960, Harada et al., 1968).

The structure of (1  3)-β-d-glucan has been studied by X-ray diffraction (Chuah et al., 1983, Deslandes et al., 1980, Marchessault et al., 1977, Takeda et al., 1978) and solid-state 13C NMR spectroscopy (Fyfe et al., 1984, Pelosi et al., 2006, Saito et al., 1981, Saito et al., 1987, Saito et al., 1989), mainly using curdlan as specimen. One reason for the interests in curdlan is its gelation phenomena in aqueous solutions (Harada et al., 1968, Marchessault et al., 1977, Saito et al., 1981, Saito et al., 1989). For crystal structure analysis, however, paramylon has important advantage of unusually high crystallinity as natural macromolecule.

Marchessault et al. (1977) identified two crystal forms of (1  3)-β-d-glucan, hydrate and anhydrous form, by X-ray diffraction of oriented fibers of curdlan prepared from dimethylsulfoxide solution. They subsequently proposed the crystal structure models of hydrate and anhydrous based on X-ray diffraction and stereochemical modeling (Chuah et al., 1983, Deslandes et al., 1980). Their model for anhydrous was a right-handed 6/1 triple helices arranged in a hexagonal unit cell with dimensions of a = 14.41 Å and c = 5.87 Å. The space group was P63, with the asymmetric unit consisting of one glucopyranoside residue, and the unit cell containing six glucose residues (Deslandes et al., 1980). Based on the structure of anhydrous, that of hydrate was determined from combination of fiber and powder X-ray diffraction as: right-handed 6/1 triple helices arranged in a hexagonal unit cell with dimensions of a = 15.56 Å and c = 18.78 Å. This structure belongs to the space group P1, with its c-axis three times longer than that of anhydrous, and is close to P3 except for the water molecules. Thus the hydrate has lower symmetry than anhydrous, and the unit cell contains 18 glucopyranoside residues and 36 water molecules (Chuah et al., 1983).

These analyses indicated the presence of one water molecules per glucose residue in the hydrate (Chuah et al., 1983). On the other hand, the number of water molecules in hydrate calculated from density measurement was two per glucose residue (Chuah et al., 1983, Marchessault et al., 1977). Chuah et al. (1983) explained this contradiction as follows: While two water molecules per glucose residue are in the crystal lattice, one is located around the O4 and O5 atoms as hydrating water, and the others are distributed randomly in the crystal. This explanation, however, remained speculative, and more precise analysis is necessary for full understanding of the hydrate structure.

For crystal structure analysis, paramylon has a disadvantage of being granules; but its high crystallinity is an alternative advantage utilized in several studies (Booy et al., 1981, Chuah et al., 1983, Kiss et al., 1988, Marchessault and Deslandes, 1979). In the present study we reexamined the crystal structures of paramylon by synchrotron X-ray powder diffraction, which allows analysis with higher precision, combined with solid-state cross-polarization/magic angle spinning (CP/MAS) 13C NMR.

Besides uncertainty in the crystal structure, there have been contradictory reports about the transition between hydrate and anhydrous forms of paramylon (Kiss et al., 1988, Marchessault and Deslandes, 1979). Marchessault and Deslandes (1979) reported that the anhydrous needs annealing at 140 °C in water to convert to hydrate. On the other hand, Kiss et al. (1988) reported that the dried samples prepared from never-dried paramylon readily converted to hydrate by immersion in water. Therefore we also studied the reversibility between the hydrate and anhydrous forms of paramylon by preparing never-dried, dried and rehydrated samples from cultured Euglena. Also the transitions by humidity change were studied by X-ray diffractometry.

Section snippets

Cultivation and preparation of paramylon sample

Euglena gracilis E-6 (IAM Culture Collection) was cultured in a medium containing 5 g of pepton, 2 g of yeast extract, 15 g of glucose, and 10 μg of cyanocobalamine in 1 L of water at 28 °C in the dark (Kitaoka, Sasaki, & Taniguchi, 1993). After cultivation for a week, the cells were collected by centrifugation (8000g, 5 min), redispersed in water, and sonicated for disrupting the cells. After centrifugation the pellet was dispersed in 0.1 M-pH 6.8 Tris–HCl buffer containing 2% sodium dodecylsulfate,

Synchrotron X-ray powder diffraction

The X-ray powder diffraction patterns of the never-dried, dried, and rehydrated samples of paramylon were recorded as in Fig. 1, Fig. 2. The native (never-dried) hydrate (Fig. 2a) gave many sharp peaks up to high Q ranges, indicating exceptionally high crystallinity. The nine peaks in Fig. 2a could be indexed according to Chuah et al. (1983). The present data allowed refinement as: a = 15.574 (1) Å, and c = 18.587 (10) Å (Table 1), closely agreeing with Chuah et al.’s values (1983).

Dehydration of the

Conclusion

The never-dried, dried, and rehydrated paramylon samples were prepared from Euglena cultured in the dark. Synchrotron X-ray powder diffraction, solid-state CP/MAS 13C NMR spectroscopy, and gravimetry analyses of the crystal structure change by hydration/dehydration lead to the following conclusions:

  • 1.

    The transitions between the hydrate and anhydrous take place reversibly. The anhydration by drying is accompanied by significant decrease in crystallinity, possibly resulting from introduction of

Acknowledgments

The synchrotron radiation experiments were performed at BL38B1 in SPring-8 with the approval of the Japan Synchrotron Research Institute (JASRI). This study was partly supported by a Grant-in-Aid for Scientific Research (No. 18780131).

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