RT Journal Article
T1 Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase co-immobilised with NADH
A1 Orrego, Alejandro
A1 Andrés-Sanz, Daniel
A1 Velasco-Lozano, Susana
A1 Sanchez-Costa, Mercedes
A1 Berenguer, José
A1 Guisán, José M.
A1 Rocha Martín, Javier
A1 López-Gallego, Fernando
AB β-Hydroxyesters are essential building blocks utilised by the pharmaceutical and food industries in the synthesis of functional products. Beyond the conventional production methods based on chemical catalysis or whole-cell synthesis, the asymmetric reduction of β-ketoesters with cell-free enzymes is gaining relevance. To this end, a novel thermophilic (S)-3-hydroxybutyryl-CoA dehydrogenase from Thermus thermophilus HB27 (Tt27-HBDH) has been expressed, purified and biochemically characterised, determining its substrate specificity towards β-ketoesters and its dependence on NADH as a cofactor. The immobilization of Tt27-HBDH on agarose macroporous beads and its subsequent coating with polyethyleneimine has been found the best strategy to increase the stability and workability of the heterogeneous biocatalyst. Furthermore, we have embedded NADH in the cationic layer attached to the porous surface of the carrier. Since Tt27-HBDH catalyses cofactor recycling through 2-propanol oxidation, we achieve a self-sufficient heterogeneous biocatalyst where NADH is available for the immobilised enzymes but its lixiviation to the reaction bulk is avoided. Taking advantage of the autofluorescence of NADH, we demonstrate the activity of the enzyme towards the immobilised cofactor through single-particle analysis. Finally, we tested the operational stability in the asymmetric reduction of β-ketoesters in batch, succeeding in the reuse of both the enzyme and the co-immobilised cofactor up to 10 reaction cycles.
PB Royal Society of Cehmistry
SN 2044-4753
SN 2044-4761
YR 2021
FD 2021
LK https://hdl.handle.net/20.500.14352/94117
UL https://hdl.handle.net/20.500.14352/94117
LA eng
NO Orrego, Alejandro H., et al. «Self-Sufficient Asymmetric Reduction of β-Ketoesters Catalysed by a Novel and Robust Thermophilic Alcohol Dehydrogenase Co-Immobilised with NADH». Catalysis Science & Technology, vol. 11, n.o 9, 2021, pp. 3217-30. https://doi.org/10.1039/D1CY00268F.
NO AgradecimientosFLG, JRM y JMG agradecen la financiación del proyecto HOTZYMES de la Unión Europea (UE) (acuerdo de subvención nº 829162) en el marco del programa H2020-FETOPEN. FLG y DAS agradecen la financiación recibida del Consejo Europeo de Investigación (ERC-Co-2018 #818089). También se agradece la financiación de IKERBASQUE a FLG. Este trabajo se realizó en el marco del Programa de Unidades de Excelencia María de Maeztu de la Agencia Estatal de Investigación de España – Subvención No. MDM-2017-0720. JB agradece la Beca Institucional de la Fundación Ramón Areces. También agradecemos el apoyo de la tasa de publicación por parte de la Iniciativa de Apoyo a las Publicaciones en Acceso Abierto del CSIC a través de su Unidad de Recursos de Información para la Investigación (URICI).
NO European Commission
NO IKERBASQUE
NO Fundación Ramón Areces
NO Consejo Superior de Investigaciones Científicas
NO Ministerio de Ciencia, Innovación y Universidades (España)
DS Docta Complutense
RD 18 abr 2025