The use of enzymes for thesynthesis of nucleoside analogues offers several advantages over multistep chemical methods,including chemo-, regio-and stereoselectivityas well asmilderreaction conditions. Herein, the production, characteri-zation and utilization of a purine nucleoside 2’-deoxyribosyl-transferase (PDT) from Trypanosoma bruceiare reported. TbPDT is a dimer which displays not only excellent activity and stability over a broad range of temperatures (50−70 ºC), pH (4−7) and ionic strength (0−500 mM NaCl) but also an unusual high stability under alkaline conditions (pH 8−10). TbPDT is shown to be proficient in the biosynthesis of numerous therapeu-tic nucleosides, including didanosine, vidarabine, cladribine, fludarabine and nelarabine. The structure-guided replacement of Val11 with either Ala or Ser resulted in variants with 2.8-fold greater activity. TbPDT was also covalently immobilized on glutaraldehyde-activated magnetic microspheres. MTbPDT3 was selected as the best derivative (4200 IU/g, activity recovery of 22%),and could be easily recaptured and recycled for >25 reac-tions with negligible loss of activity. Finally, MTbPDT3 was suc-cessfully employed in the expedient synthesisof several nucleo-side analogues. Taken together,our results support the notion thatTbPDT has good potentialas an industrial biocatalyst for the synthesis of a wide range of therapeutic nucleosides through an efficient and environmentally friendly methodology.