60 MICROBIAL CELL WALLS 



was suggested that the "disaccharide" formed an important 

 structural unit of the cell-wall mucocomplex.-° Additional 

 products reacting more weakly with various spray reagents 

 were detectable in the dialyzable fractions, but their nature 

 remained unknown until their recent isolation and charac- 

 terization.^^-^ 



In our investigation in 1959 we were able to confirm the 

 nature of the disaccharide and suggest the structure of this 

 compound. The isolated disaccharides from Micrococcus 

 lysodeikticus walls digested with egg-white lysozyme and 

 Streptomyces Fj enzyme were investigated by reaction of the 

 compounds with NaBH4 and by degradation with /?-gluco- 

 sidase. The products of reaction of the compounds with 

 NaBH4 clearly established the identity of the reducing 

 group liberated by lysozyme action as that of muramic acid, 

 thus providing direct experimental evidence for the hypo- 

 thetical structure of the lysozyme substrate proposed by 

 Brumfitt, Wardlaw, and Park. 22 The breakdown of the 

 disaccharides into the free N-acetylamino sugars, N-acetyl- 

 glucosamine and N-acetylmuramic acid, provided evidence 

 of the ^-glycosidic bond. The structure of the disaccharide 

 and the nature of the products formed from reaction with 

 NaBH4 and yg-glucosidase are presented in Fig. 12. The 

 presence of a 1-^6 linkage was suggested from experi- 

 ments performed on [^^C] disaccharide oxidized with NaI04 

 and determining the recovery of [^*C] formaldehyde.^^, 23 



In addition to the disaccharide, an oligosaccharide yield- 

 ing glucosamine and muramic acid on hydrolysis was de- 

 tected, and its structure investigated, by the techniques used 

 in studying the disaccharide. That the compound was a 

 tetrasaccharide was supported by measuring the ratios of 

 glucosamine, muramic acid, and "muramicitol" (the amino 

 sugar hexitol of muramic acid ^2,23^ separated after hydrol- 

 ysis of the substance reduced with NaBH4. Both lysozyme 



