An active d-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as d-PfHYD. Sequence and consensus analysis suggests that d-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. d-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. d-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg(-1). d-PfHYD showed the highest activity at pH 9.0 and 60 A degrees C. Metal ions such as Mn2+, Fe2+, and Fe3+ could activate d-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified d-PfHYD preferred aliphatic to aromatic 5'-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of d-PfHYD. Employing 1.0 g L-1 recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into d-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant d-PfHYD could be potentially applied in the synthesis of d-amino acids.