Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

摘要

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100,000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degreesC, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu2+ and Hg2+. No effect on enzyme activity was detected with any other divalent cations. EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings. (C) 2004 Elsevier SAS. All rights reserved. [References: 19]