Self-sufficient asymmetric reduction of β-ketoesters catalysed by a novel and robust thermophilic alcohol dehydrogenase coimmobilised with NADH

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 singleparticle
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.