Enzymatic Discrimination of 2‐Acetamido‐2‐deoxy‐ D ‐mannopyranose‐Containing Disaccharides Using β‐ N ‐Acetylhexosaminidases

β‐ N ‐Acetylhexosaminidase from Aspergillus oryzae selectively discriminates mixture of the disaccharides GlcNAcβ(1→4)GlcNAc ( 1 ) and GlcNAcβ(1→4)ManNAc ( 2 ). N,N′ ‐Diacetylchitobiose ( 1 ) was selectively hydrolyzed by β‐ N ‐acetylhexosaminidase, whereas its C‐2 epimer ( 2 ) was completely resistant to the enzyme hydrolysis. Analogous discrimination was observed also with GalNAcβ(1→4)GlcNAc ( 3 ) and GalNAcβ(1→4)ManNAc ( 4 ). β‐ N ‐Acetylhexosaminidases from A. terreus, A. flavus , bovine kidney and bovine epididymis displayed the same selectivity, whereas the enzymes from A. sojae, A. tamarii, Penicillium brasilianum, P. oxalicum, P. funiculosum, P. multicolor, Talaromyces flavus and jack beans hydrolyzed both types of disaccharides. Molecular modelling of β‐ N ‐acetylhexosaminidase from A. oryzae CCF 1066 and docking experiments with both types of disaccharides revealed that the ManNAc residue causes distortion of disaccharide molecule resulting in a steric conflict with a Trp 482 that causes diminished stabilization of the oxazolinium transition state by extending the distance of Asp 345 in the active site. Both ManNAc‐containing disaccharides 2 and 4 dock with similar steric energies into the active site but without cleaving and also without notable inhibition. This novel phenomenon enables also the preparative production of both disaccharides 2 and 4 starting from N, N '‐diacetylchitobiose ( 1 ) or GalNAcβ(1→4)GlcNAc ( 3 ) followed by Lobry de Bruyn–Albreda van Ekenstein C‐2 epimerization catalyzed by Ca(OH) 2 . The resultant mixture of the respective epimers 1, 2 or 3, 4 that is hardly separable by, e.g., analytical HPLC can be treated with the β‐ N ‐acetylhexosaminidase from A. oryzae and the mixture of monosaccharides and target disaccharide can be easily separated using gel filtration.