Enzymatic discrimination of 2-acetamido-2-deoxy-D-mannopyranose-containing disaccharides using β-N-acetylhexosaminidases

beta-N-Acetylhexosaminidase from Aspergillus oryzae selectively discriminates mixture of the disaccharides GlcNAcbeta(1-->4)GlcNAc (1) and GlcNAcbeta(1-->4)ManNAc (2). N,N'-Diacetylchitobiose (1) was selectively hydrolyzed by beta-N-acetylhexosaminidase, whereas its C-2 epimer (2) was completely resistant to the enzyme hydrolysis. Analogous discrimination was observed also with GalNAcbeta(1-->4)GlcNAc (3) and GalNAcbeta(1-->4)ManNAc (4). beta-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 beta-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 GalNAcbeta(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 beta-N-acetylhexosaminidase from A. oryzae and the mixture of monosaccharides and target disaccharide can be easily separated using gel filtration.