期刊
CATALYSTS
卷 12, 期 5, 页码 -出版社
MDPI
DOI: 10.3390/catal12050474
关键词
anomer separation; N-acetylhexosaminidase; glycoside; chromogenic enzyme substrates; enzyme immobilization
资金
- Agency for supporting research and development [APVV-20-0208]
A new method combining chemical synthesis and enzymatic hydrolysis was developed to prepare glycoside substrates with high selectivity. The immobilization of Hex in polyvinyl alcohol hydrogel capsules showed improved stability and retained high activity.
alpha-Nitrophenyl derivatives of glycosides are convenient substrates used to detect and characterize alpha-N-acetylgalactosaminidase. A new procedure combining chemical and biocatalytic steps was developed to prepare 4-nitrophenyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (4NP-alpha-GalNAc). The alpha-anomer was prepared through chemical synthesis of an anomeric mixture followed by selective removal of the beta-anomer using specific enzymatic hydrolysis. Fungal beta-N-acetylhexosaminidase (Hex) from Penicillium oxalicum CCF 1959 served this purpose owing to its high chemo-and regioselectivity towards the beta-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 degrees 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.
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