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Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2'-deoxyribosyltransferase from Leishmania mexicana ( Lm PDT) and Escherichia coli hypoxanthine phosphoribosyltransferase ( Ec HPRT) were immobilized onto Ni 2+ -prechelated magnetic microspheres (MagReSyn ® NTA). Among the resulting derivatives, M Lm PDT3 (activity: 11,935 IU/g support , 63% retained activity, operational conditions: 40 °C and pH 5-7) and M Ec HPRT3 (12,840 IU/g support , 45% retained activity, operational conditions: pH 5-8 and 40-60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the M Lm PDT3/M Ec HPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to M Lm PDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, M Lm PDT3/M Ec HPRT3 was reused for 16 cycles, which displayed a 75% retained activity.

One of the major drawbacks of the industrial implementation of enzymatic processes is the low operational stability of the enzymes under tough industrial conditions. In this respect, the use of thermostable enzymes in the industry is gaining ground during the last decades. Herein, we report a structure-guided approach for the development of novel and thermostable 2′-deoxyribosyltransferases (NDTs) based on the computational design of disulfide bonds on hot spot positions. To this end, a small library of NDT variants from Lactobacillus delbrueckii (LdNDT) with introduced cysteine pairs was created. Among them, LdNDTS104C (100% retained activity) was chosen as the most thermostable variant, displaying a six- and two-fold enhanced long-term stability when stored at 55 °C (t1/255 °C ≈ 24 h) and 60 °C (t1/260 °C ≈ 4 h), respectively. Moreover, the biochemical characterization revealed that LdNDTS104C showed >60% relative activity across a broad range of temperature (30−90 °C) and pH (5−7). Finally, to study the potential application of LdNDTS104C as an industrial catalyst, the enzymatic synthesis of nelarabine was successfully carried out under different substrate conditions (1:1 and 3:1) at different reaction times. Under these experimental conditions, the production of nelarabine was increased up to 2.8-fold (72% conversion) compared with wild-type LdNDT.