Enzymatic reduction of levulinic acid (LA) was performed for the synthesis of 4-hydroxyvaleric acid (4HV) - a monomer of bio-polyester and a precursor of bio-fuels - using 3-hydroxybutyrate dehydrogenase (3HBDH) from Alcaligenes faecalis. Due to the catalytic inactivity of the wild-type enzyme toward LA, engineering of the substrate specificity of the enzyme was performed. A rational design approach with molecular docking simulation was applied, and a double mutant, His144Leu/Trp187Phe, which has catalytic activity (k(cat)/K-m = 578.0 min(-1) M-1) toward LA was generated. Approximately 57% conversion of LA to 4HV was achieved with this double mutant in 24 h, while no conversion was achieved with the wildtype enzyme. (C) 2013 Elsevier Ltd. All rights reserved.