In-vitro-Studien zur destabilisierenden Wirkung lyophilisierter Saccharomyces cerevisiae Hansen CBS 5926-Zellen auf Enterobakterien. Läβt sich diese Eigenschaft biochemisch erklären?

A large number of linear and branched oligosaccharides and several glycosides of D-mannose were tested for their inhibitory activity on the agglutination of yeast cells or guinea pig erythrocytes by three D-mannose-specific enteric bacteria possessing type 1 fimbriae. With Escherichia coli 346, the best inhibitors found are the alpha glycosides of the branched oligosaccharides alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp-(1 leads to 6)-alpha-D-Manp-(1 leads to 3)-D-Manp and alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-alpha-D-Manp- (1 leads to 6)-[alpha-D-Manp-(1 leads to 2)-alpha-D-Manp-(1 leads to 3) ]-D-Manp and the trisaccharide alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc, all of which are 21-30 times more inhibitory than methyl alpha-D-mannopyranoside. The aromatic glycoside p-nitrophenyl alpha-D-mannopyranoside was also a strong inhibitor (30 times more inhibitory than methyl alpha-D-mannopyranoside), whereas the corresponding beta-D-glycoside was only a weak inhibitor (approximately as methyl alpha-D-mannopyranoside). A nearly identical pattern of inhibitory activity was observed with the fimbriae. This suggests that the combining site of the E. coli fimbrial lectin is in the form of an extended pocket on the surface of the lectin corresponding to the size of a trisaccharide and fitting best the structure alpha-D-Manp-(1 leads to 3)-beta-D-Manp-(1 leads to 4)-D-GlcNAc. Since p-nitrophenyl alpha-D-mannopyranoside is a strong inhibitor, the existence of a hydrophobic region in the combining site or close to it was assumed. The combining site of the Klebsiella pneumoniae fimbrial lectin is probably similar to that of E. coli, but that of the Salmonella typhimurium fimbrial lectin differs considerably. It appears that the combining sites of the three bacterial lectins tested exhibit preference for structures found in N-glycosylic oligomannoside units of mammalian cell surface glycoproteins.

Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a beta-1,3-glucanase with a molecular weight of 55,000. The K(m) for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of beta-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.