Cruz, GuillermoSaiz, Laura PilarBilal, MuhammadEltoukhy, LobnaLoderer, ChristophFernández Lucas, Jesús2024-10-102024-10-102022-11Cruz, Guillermo, et al. «Magnetic Multi-Enzymatic System for Cladribine Manufacturing». International Journal of Molecular Sciences, vol. 23, n.o 21, noviembre de 2022, p. 13634. DOI.org (Crossref), https://doi.org/10.3390/ijms232113634.1422-006710.3390/ijms232113634https://hdl.handle.net/20.500.14352/108867Enzyme-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 (LmPDT) and Escherichia coli hypoxanthine phosphoribosyltransferase (EcHPRT) were immobilized onto Ni2+-prechelated magnetic microspheres (MagReSyn®NTA). Among the resulting derivatives, MLmPDT3 (activity: 11,935 IU/gsupport, 63% retained activity, operational conditions: 40 °C and pH 5–7) and MEcHPRT3 (12,840 IU/gsupport, 45% retained activity, operational conditions: pH 5–8 and 40–60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MLmPDT3/MEcHPRT3 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 MLmPDT3), 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, MLmPDT3/MEcHPRT3 was reused for 16 cycles, which displayed a 75% retained activity.engAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/journal articlehttps://doi.org/10.3390/ijms232113634https://www.mdpi.com/1422-0067/23/21/13634open access577.15577.260661.12Cascade synthesisMagnetic catalystsEnzyme immobilizationTransglycosylation reactionNucleoside analoguesBioquímica (Biología)Biología molecular (Biología)BiotecnologíaMedicamentos2403 Bioquímica2415 Biología Molecular2302.09 Enzimología3302 Tecnología Bioquímica3209.01 Análisis de Medicamentos