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 32,000 Da 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 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), 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.
Las Heras-Vázquez
Francisco Javier
FJ
Departamento de Química-Física, Bioquímica y Química Inorgánica. Edificio C.I.T.E.I. Universidad de Almería., La Cañada de San Urbano, Spain E-04120.
Martínez-Rodríguez
Sergio
S
Mingorance-Cazorla
Lydia
L
Clemente-Jiménez
Josefa María
JM
Rodríguez-Vico
Felipe
F
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D019998:Racemases and Epimerases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D011994:Recombinant Proteins; D019998:Racemases and Epimerases; C074773:hydantoin racemase