Stabilization of d-hydantoinase by intersubunit cross-linking

It was observed that tetrameric d-hydantoinase from Bacillus stearothermophilus SD1 is dissociated into monomers under operational conditions, resulting in a detrimental loss of its catalytic activity. As an approach to reduce the dissociation of subunits and to maintain its catalytic activity, intersubunit cross-linking was attempted by using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride). The cross-linking conditions were optimized in terms of stabilization and catalytic activity of the recovered enzyme. Cross-linked d-hydantoinase showed a four-fold longer half-life under operational conditions and was very stable even at an elevated temperature, whereas the native enzyme was almost completely deactivated. In addition, intersubunit cross-linking of d-hydantoinase also led to stabilization of the enzyme in the presence of 20% methanol and under acidic conditions. The cross-linked enzyme was more efficient in the conversion of substrate, which seems to be due to the increased stability of enzyme.