We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Gal beta 1-3GalNAc) from core1-pNP (Gal beta 1-3GalNAc alpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Gal beta 1-3[GlcNAc beta 1-6]GalNAc alpha-pNP) and tetrasaccharides from Gal- core2-pNP (Gal beta 1-3[Gal beta 1-3Glc-NAc beta 1-6]GalNAc alpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research. (C) 2008 Elsevier Inc. All rights reserved.