α-Nitrophenyl derivatives of glycosides are convenient substrates used to detect and characterize α-N-acetylgalactosaminidase. A new procedure combining chemical and biocatalytic steps was developed to prepare 4-nitrophenyl-2-acetamido-2-deoxy-α-D-galactopyranoside (4NP-α-GalNAc). The α-anomer was prepared through chemical synthesis of an anomeric mixture followed by selective removal of the β-anomer using specific enzymatic hydrolysis. Fungal β-N-acetylhexosaminidase (Hex) from Penicillium

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... icum CCF 1959 served this purpose owing to its high chemo-and regioselectivity towards the β-anomeric N-acetylgalactosamine (GalNAc) derivative. The kinetic measurements of the hydrolytic reaction showed that the enzyme was not inhibited by the substrate or reaction products. The immobilization of Hex in lens-shaped polyvinyl alcohol hydrogel capsules provided a biocatalyst with very good storage and operational stability. The immobilized Hex retained 97% of the initial activity after ten repeated uses and 90% of the initial activity after 18 months of storage at 4 °C. Immobilization inactivated 65% of the enzyme activity. However, the effectiveness factor and kinetic and mass transfer phenomena approached unity indicating negligible mass transfer limitations.