Immobilization of thermostable nucleoside phosphorylases on MagReSyn® epoxide microspheres and their application for the synthesis of 2,6-dihalogenated purine nucleosides

• Nucleoside phosphorylases were efficiently immobilized on the magnetic beads.
• Key factors for immobilization were found by a DoE approach.
• The immobilized enzymes were successfully applied for the synthesis of modified nucleosides.
• An interesting finding consists of the increasing of the enzymatic activity upon storage.

Immobilization of enzymes has been considered as an efficient approach to facilitate enzyme recovery and to improve biocatalyst stability. However, multimeric nucleoside phosphorylases, useful for the synthesis of modified nucleosides, encounter several challenges to their immobilization, including requirement for high enzymatic load, and poor retention of enzyme activity. In this study, multimeric enzymes pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase (PNP), from Thermus thermophilus and Geobacillus thermoglucosidasius, respectively, were successfully immobilized on the magnetic beads with cross-linked polyethyleneimine and epoxide functional groups, resulting in high enzyme loading (up to 0.4 and 1.3 g per g dry beads), high enzyme activity maintenance (41% and 83% under the highest enzyme loading), and improved enzyme stability. The screening of the immobilization conditions showed that binding buffer pH, enzyme loading amount, binding temperature and binding time are important factors for the immobilization yield. The application of the immobilized enzymes in the synthesis of 2,6-dihalogenated purine nucleosides achieved with high substrate conversion (78.5–85.5%) as well as high productivity (1.5–2.0 g L−1 h−1).

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