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 oxalicum 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.

中文翻译：

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4-硝基苯基-2-乙酰氨基-2-脱氧-α-D-吡喃半乳糖苷的化学酶促生产使用固定化β-N-乙酰氨基己糖苷酶

糖苷的 α-硝基苯基衍生物是用于检测和表征 α- N-乙酰半乳糖胺酶的方便底物。开发了一种结合化学和生物催化步骤的新工艺来制备 4-nitrophenyl-2-acetamido-2-deoxy-α-D-galactopyranoside (4NP-α-GalNAc)。α-异头物通过异头混合物的化学合成制备，然后使用特定的酶水解选择性去除 β-异头物。来自草酸青霉CCF 1959 的真菌 β- N-乙酰氨基己糖苷酶 (Hex)因其对 β-异头N的高化学和区域选择性而发挥了这一作用-乙酰半乳糖胺（GalNAc）衍生物。水解反应的动力学测量表明酶不受底物或反应产物的抑制。Hex在透镜状聚乙烯醇水凝胶胶囊中的固定化提供了一种具有非常好的储存和操作稳定性的生物催化剂。固定的 Hex 在 10 次重复使用后保留了 97% 的初始活性，在 4°C 下储存 18 个月后保留了 90% 的初始活性。固定化使 65% 的酶活性失活。然而，有效性因子以及动力学和传质现象接近统一，表明传质限制可忽略不计。