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

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 (Me 2SO) 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.