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

Abstract Extracellular β- N -acetylhexosaminidases from different fungal strains were screened for their ability to synthesize nucleotide-activated oligosaccharides. In combination with GalNAc(β1- p NP) 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- p NP) in aqueous solutions at room temperature. HM-β-CD also increased the maximum reaction velocity ( V max ) of the enzyme, which was probably due to the elimination of enzyme inhibitors, e.g., p NP 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 1 H and 13 C NMR spectroscopy and MALDI-TOF mass spectrometry.