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

Extracellular beta-N-acetylhexosaminidases from different fungal strains were screened for their ability to synthesize nucleotide-activated oligosaccharides. In combination with GaINAc(beta1-pNP) as donor the nucleotide sugar UDP-GlcNAc was found to be an effective acceptor substrate for beta-N-acetylhexosaminidases from Aspergillus parasiticus, Aspergillus flavus, Penicillium oxalicum, Trichoderma harzianum, Aspergillus flavipes, Aspergillus tamarii, and Aspergillus oryzae. beta-N-acetylhexosaminidase from Trichoderma harzianum was selected for further studies on the synthesis of the UDP-disaccharide GalNAc(beta1-4)GlcNAc(alpha1-UDP) (UDP-LacdiNAc). The addition of heptakis-(2,6-di-O-methyl)-beta-cyclodextrin (HM-beta-CD) was crucial for the synthesis of this compound by increasing the solubility of the donor substrate GaINAc(beta1-pNP) in aqueous solutions at room temperature. HM-beta-CD also increased the maximum reaction velocity (V-max) of the enzyme, which was probably due to the elimination of enzyme inhibitors, e.g., pNP and/or GaINAc, by the cyclodextrin derivative. Under optimized conditions GaINAc(beta1-4)GlcNAc(alpha1-UDP) was formed stereo- and regioselectively with an overall yield of 3.5% (17.7 mumol, 15.1 mg). The chemical structure was characterized by H-1 and C-13 NMR spectroscopy and MALDI-TOF mass spectrometry. (C) 2004 Elsevier Inc. All rights reserved.