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00925njm 22002777a 4500 dc Cruz, Guillermo author Acosta, Javier author Mancheño Gómez, José Miguel author Del Arco, Jon author Fernández Lucas, Jesús author 2022 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. Cruz, Guillermo, et al. «Rational Design of a Thermostable 2′-Deoxyribosyltransferase for Nelarabine Production by Prediction of Disulfide Bond Engineering Sites». International Journal of Molecular Sciences, vol. 23, n.o 19, octubre de 2022, p. 11806. DOI.org (Crossref), https://doi.org/10.3390/ijms231911806. 1661-6596 10.3390/ijms231911806 https://hdl.handle.net/20.500.14352/108897 1422-0067 https://doi.org/10.3390/ijms231911806 https://www.mdpi.com/1422-0067/23/19/11806 Rational design of a thermostable 2′-deoxyribosyltransferase for nelarabine production by prediction of disulfide bond engineering sites