Synthesis in vitro of crystalline chitin by a solubilized enzyme from the cellulosic fungus Saprolegnia monoica

Enriched preparations of chitin synthase were obtained from cell homogenates from Saprolegnia monoica. Chitin synthase was solubilized from a mixed membrane fraction by two successive digitonin treatments. Glycerol gradient centrifugation of the solubilized proteins separated the chitin synthase activity from the majority of proteins and from β-1,3 and β-1,4 glucan synthases. The properties of chitin synthase from this Oomycete fungus are similar to those reported for the enzymes of chitinous fungi. The solubilized enzyme catalysed the synthesis in vitro of spindle-like crystals of chitin. Biophysical analysis (by electron and X-ray diffractometry and infrared spectroscopy) demonstrated that the polymer synthesized in vitro was α-chitin. These results and those previously reported demonstrate unambiguously that chitin synthase and chitin are normal components of the cell wall of the cellulosic fungus S. monoica.