A Refined Method for the Determination of Saccharomyces cerevisiae Cell Wall Composition and β-1,6-Glucan Fine Structure

摘要: In yeast and other fungi, cell division, shape, growth depend on the coordinated synthesis degradation of wall polymers. We have developed a reliable efficient micro method to determine Saccharomyces cerevisiae composition that distinguishes between beta1,3- beta1,6-glucan. The is based sequential treatment walls with specific hydrolytic enzymes followed by dialysis. low molecular weight (MW) products thus separated account for each particular polymer. can be applied as little 50-100 mg (wet wt) radioactively labeled cells. A combination chitinase recombinant beta-1,3-glucanase initially used, releasing all chitin 60-65% beta1,3-glucan from walls. Next, endo-beta-1,6-glucanase Trichoderma harzianum utilized release beta-1,6-glucan present in wall. chromatographic pattern endoglucanase digested provides characteristic "fingerprint" fine structure oligosaccharides this was determined 1H NMR electrospray ionization mass spectroscopy. final enzymatic step uses laminarinase beta-glucosidase remaining beta-1,3-glucan. mannan remains high MW fraction at end fractionation procedure. Good sensitivity correlation traditional methods were observed wild-type several mutants.