Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58
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Las Heras Vázquez, Francisco Javier; Martínez Rodríguez, Sergio; Mingorance Cazorla, Lydia; Clemente Jiménez, Josefa María; Rodríguez Vico, FelipeDate
2003Abstract
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 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal
conditions for hydantoin racemase activity were pH 7.5 and 55 C with L-5-ethylhydantoin as substrate. Enzyme activity was
slightly affected by the addition of Ni2þ and Co2þ and strongly inhibited by Cu2þ and Hg2þ. No effect on enzyme activity was
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Palabra/s clave
Racemization
D-Amino acid
Hydantoin racemase
Purification