To recover active DCaseH, a recombinant N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-His peptide, from Escherichia coli inclusion bodies, a dilution-additive-type refolding strategy was employed for both urea-solubilized inclusion solutions and. purified denatured DCaseH. All refoldings of the inclusion solutions led to an increase (blue shift) in the average emission energy of the intrinsic tryptophan fluorescence, while only the ones with the addition of 30-50% glycerol effectively restored DCaseH activity, with a best activity achieved at 40 mu g/mL total protein concentration, 50% glycerol, and pH 8 for 22 days. A further native purification using metal affinity resins yielded pure DCaseH of 154 U/mg, which is 37% of the native activity. On the other hand, the refolding of the purified denatured DCaseH was best yielded at 50% glycerol for 3 days, with a 20% renaturation yield. Circular dichroism analyses of the refolded pure DCaseH revealed that approximately 38%. of the native secondary structure was reformed. Therefore, the refolding with the addition of glycerol partially recovered both the activity and the native-like secondary structure of denatured DCaseH. As for better renaturation, a refolding of solubilized inclusion solutions followed by a native purification is suggested. (c) 2004 Elsevier Ltd. All rights reserved.