Diplococcal beta-galactosidase with a specificity reacting to beta 1-4 linkage but not to beta 1-3 linkage as a useful exoglycosidase for the structural elucidation of glycolipids.

Diplococcal beta-galactosidase, which is known to be useful for the structural studies of glycoprotein-linked oligosaccharides, was found to show the same substrate specificity in cleaving Gal beta 1-4 linkages of glycolipids as that of the oligosaccharides. The optimum conditions of beta-galactosidase in the 80% ammonium sulfate precipitates of the culture medium of Streptococcus (Diplococcus) pneumoniae were determined with nLcOse4Cer radiolabeled by the galactose oxidase-NaB3H4 procedure. Detergent was required for the highest activity, and different combinations of several buffers and detergents showed different properties in stimulating beta-galactosidase, and in enhancing or suppressing N-acetyl-beta-hexosaminidase which was contaminated in the enzyme preparation. The optimum pH was found to be at 6.5, and specific activity and Km were 8.1 nmol/mg protein/h and 1 nmol, respectively. While more than 70% of beta-galactose was liberated from LacCer and nLcOse4Cer within 1 h under the optimum conditions to form GlcCer and nLcOse3Cer, respectively, none was liberated from LcOse4Cer, GalCer, GgOse4Cer, GbOse3Cer, IV3 alpha GalnLcOse4Cer, and Il3NeuAcGgOse4Cer, showing the substrate specificity solely to Gal beta 1-4 linkage.