Purification and biochemical characterization of D-hydantoinase and D-N-carbamoylase from Burkholderia cepecia.njut01

Hydantoinase and N-carbamoylase play important roles in the production of optically pure amino acids from racemic 5-mono-substituted hydantoins. In this report, hydantoinase and the N-carbamoylase from Burkholderia cepecia.njut01 were purified to homogeneity by chromatography (Pharmacia Explorer 100 system). The substrate specificity, enantioselectivity, pH dependence of activity and temperature stability of the activity were characterized. The results show that the hydantoinase and N-carbamoylase induced from Burkholderia cepecia.njut01 are both strict D-stereo selective enzymes. They both hydrolyze substrates with side chains containing aliphatic and aromatic residues with higher activity and affinity toward aromatic than aliphatic substituted substrates. The hydantoinase is a homotetramer with subunit molecular weight near 52,000 and is active between pH 6.5 and 10 with an optimum near pH 9.0. The enzyme is active at temperatures up to 60°C, however, it appears instable at higher temperatures. The subunit molecular weight of N-carbamoylase is about 35KD. The N-carbamoylase is active in the pH range from 6.0 to 9.5. The optim-pH is 7.2 and the optimizing bioconversion temperature of the N-carbamyolase is 52°C.