Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55degreesC with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni2+ and Co2+ and strongly inhibited by Cu2+ and Hg2+. No effect on enzyme activity was detected with Mn2+, EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings. (C) 2003 Elsevier Science (USA). All rights reserved.