Solubilization of yeast cell-wall β-(1→3)-d-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction

doi:10.1016/S0008-6215(99)00049-X

Carbohydrate Research

Volume 316, Issues 1–4, 15 March 1999, Pages 161-172

https://doi.org/10.1016/S0008-6215(99)00049-X Get rights and content

Abstract

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)-d-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)-d-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me₂SO) extraction (yield of 9.6±4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)-d-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10⁻¹¹ g/mL.

Section snippets

. Introduction

Candida spp. is a medically important genus of fungi that induces disseminated candidiasis and candidemia in hospitalized, immunocompromised patients. The cell wall of Candida is mainly composed of two polysaccharides, mannan and β-glucan, and at least a part of the β-glucan, mainly the β-(1→3)-d-glucan moiety, is basically insoluble in H₂O or NaOH and is quite difficult to extract [1], [2]. To analyse the architecture of the yeast cell-wall, traditional procedures, such as repeated acid and/or

Materials

All strains of Candida albicans, Candida parapsilosis, and Saccharomyces cerevisiae were purchased from the Institute for Fermentation, Osaka, maintained on Sabouraud agar (Difco, USA) at 25 °C and transferred once every 3 months. Sodium hypochlorite solution and sodium hydroxide were purchased from Wako Pure Chemical Industries, Ltd. The limulus G-test (Fungitec G test MK) and zymolyase (20T and 100T) were from Seikagaku Corp. (Tokyo), and distilled water (DIW) was from Otsuka Co., Ltd.

Preparation of cell-wall β-glucan by NaClO oxidation, followed by Me₂SO extraction

Acetone-dried yeast cells were suspended in 0.1 M NaOH and oxidized with NaClO solution for 1 day at 4 °C. After the reaction was completed, the reaction mixture was centrifuged to collect a particulate substance, and the residue was washed extensively with water and dried with ethanol and acetone. During establishment of the preparation procedure, dialysis of the whole reaction mixture was performed instead of centrifugation. Components and yields were comparable in either method; thus we chose

. Discussion

We established a convenient, two-step, procedure to solubilize the yeast cell-wall β-(1→3)-d-glucan by combined use of NaClO oxidation and Me₂SO extraction. This method was applied to several strains of Candida and Saccharomyces. The structures of all the products were essentially confirmed as β-(1→3)-d-glucan, but covalently bound with various amounts of the β-(1→6)-d-glucan moiety. The proportion of the β-(1→6)-d-glucan moiety was lower in examples submitted to harsher NaClO oxidation. The

Acknowledgements

The authors would like to express sincere thanks to Mr Y. Ohgoshi for excellent technical assistance.

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Solubilization of yeast cell-wall β-(1→3)-d-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction

Abstract

Section snippets

. Introduction

Materials

Preparation of cell-wall β-glucan by NaClO oxidation, followed by Me2SO extraction

. Discussion

Acknowledgements

Arch. Biochem. Biophys.

Carbohydr. Res.

Carbohydr. Res.

FEMS Immunol. Med. Microb.

Carbohydr. Res.

Carbohydr. Res.

FEMS Immunol. Med. Microb.

Lancet

Can. J. Chem.

J. Gen. Microbiol.

Microbiol. Immunol.

Biol. Pharm. Bull.

Biol. Pharm. Bull.

Preparation of cell-wall β-glucan by NaClO oxidation, followed by Me₂SO extraction