Immobilized Nucleoside\n2′-Deoxyribosyltransferases\nfrom Extremophiles for Nucleoside Biocatalysis

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