Production, IMAC Purification, and Molecular Modeling of N-Carbamoyl-d-amino Acid Amidohydrolase C-Terminally Fused with a Six-His Peptide

A six‐His peptide was genetically engineered to the C‐terminus of Agrobacterium radiobacter N‐carbamoyl‐d‐amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one‐step IMAC‐purified. The production study showed that DCaseH was optimally produced at 15 °C for 25 h by the induction of 0.05 mM IPTG. Both Co2+‐chelated TANOL gels and Ni2+‐chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11‐fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid‐state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 °C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six‐His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.