Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids.
Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.
Martínez-Gómez
Ana Isabel
AI
Departamento de Química-Física, Bioquímica y Química Inorgánica, Edificio C.I.T.E.I., Universidad de Almería, La Cañada de San Urbano, Almería E-04120, Spain.
Martínez-Rodríguez
Sergio
S
Clemente-Jiménez
Josefa María
JM
Pozo-Dengra
Joaquín
J
Rodríguez-Vico
Felipe
F
Las Heras-Vázquez
Francisco Javier
FJ
D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000596:Amino Acids / Q000096:biosynthesis* / Q000737:chemistry; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D013237:Stereoisomerism
D000596:Amino Acids; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C020047:dihydropyrimidinase; D019998:Racemases and Epimerases; C074773:hydantoin racemase