Immobilized Nucleoside 2′-Deoxyribosyltransferases from Extremophiles for Nucleoside Biocatalysis

The synthesis of nucleosides is crucial for pharmaceutical and biotechnological applications, acting as drugs and as essential building blocks for numerous therapeutic agents. However, most enzymes employed in nucleoside biocatalysis are not recycled, possess limited stability, and have strict substrate selection for ribonucleosides or 2'deoxyribonucleosides. We employed 2'-deoxyribonucleoside transferase (NDT) enzymes from thermophilic and psychrophilic bacteria to demonstrate they can be immobilized to enhance specific activity, stability, and recyclability. NDT enzymes from <i>Chroococcidiopsis thermalis</i> (<i>Ct</i>NDT), and <i>Bacillus psychrosaccharolyticus</i> (<i>Bp</i>NDT) were immobilized by covalent attachment to chitosan beads. A double mutant of <i>Ct</i>NDT, capable of generating 3'deoxyribonucleosides, showed remarkable and increased stability after immobilization compared to the same enzyme in the solution. Furthermore, we demonstrated the recyclability of immobilized biocatalysts, with a 10-fold improvement in reaction yield over 20 consecutive cycles, highlighting the practicality and sustainability of the developed immobilization method. We used our strategy to produce a pharmaceutically relevant 3'deoxyribonucleoside (2-fluoro-3'-deoxyadenosine). This highlights the importance of efficient immobilization techniques to enhance the catalytic properties of NDT enzymes, expanding their utility in biocatalysis.