Screening and characterization of β-N-acetylhexosaminidases for the synthesis of nucleotide-activated disaccharides

Extracellular β-N-acetylhexosaminidases from different fungal strains were screened for their ability to synthesize nucleotide-activated oligosaccharides. In combination with GalNAc(β1-pNP) as donor the nucleotide sugar UDP-GlcNAc was found to be an effective acceptor substrate for β-N-acetylhexosaminidases from Aspergillus parasiticus, Aspergillus flavus, Penicillium oxalicum, Trichoderma harzianum, Aspergillus flavipes, Aspergillus tamarii, and Aspergillus oryzae. β-N-acetylhexosaminidase from Trichoderma harzianum was selected for further studies on the synthesis of the UDP-disaccharide GalNAc(β1–4)GlcNAc(α1-UDP) (UDP-LacdiNAc). The addition of heptakis-(2,6-di-O-methyl)-β-cyclodextrin (HM-β-CD) was crucial for the synthesis of this compound by increasing the solubility of the donor substrate GalNAc(β1-pNP) in aqueous solutions at room temperature. HM-β-CD also increased the maximum reaction velocity (Vmax) of the enzyme, which was probably due to the elimination of enzyme inhibitors, e.g., pNP and/or GalNAc, by the cyclodextrin derivative. Under optimized conditions GalNAc(β1–4)GlcNAc(α1-UDP) was formed stereo- and regioselectively with an overall yield of 3.5% (17.7 μmol, 15.1 mg). The chemical structure was characterized by 1H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry.