Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli.
A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.
Martinez-Rodriguez
Sergio
S
Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio CITE I, Universidad de Almería, La Cañada de San Urbano E-04120, Spain.
Las Heras-Vazquez
Francisco Javier
FJ
Clemente-Jimenez
Josefa María
JM
Mingorance-Cazorla
Lydia
L
Rodriguez-Vico
Felipe
F
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006827:Hydantoins / Q000378:metabolism*; D008715:Methionine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D019998:Racemases and Epimerases / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology; D013237:Stereoisomerism; D013379:Substrate Specificity
D000596:Amino Acids; D006827:Hydantoins; D008715:Methionine; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C020047:dihydropyrimidinase; D019998:Racemases and Epimerases; C074773:hydantoin racemase