title: Cloning and overexpression of thermostable D-hydantoinase from thermophile in E. coli and its application to the synthesis of optically active D-amino acids.
abstract: We cloned the thermostable D-hydantoinase gene from B. stearothermophilus SD-1 into E. coli. The cloned gene was constitutively expressed by its own promoter, and the enzyme was produced in its soluble form. The specific activity of the recombinant E. coli was 30 times higher than that of B. stearothermophilus SD-1. The cultivation conditions were investigated for the overproduction of the enzyme, and the temperature was found to affect the plasmid content and the expression level of the enzyme. Recombinant E. coli was cultivated in 30-L batch fermentation, the cell concentration reached 25 g-DCW/L, and the specific activity was about 20,000 units/g-DCW. D-Hydantoinase produced from the recombinant E. coli could be successfully applied to the synthesis of N-carbamoyl-D-amino acid from the 5-monosubstituted hydantoin derivative.
authors.affiliation: Department of Biotechnology, Korea Advanced Institute of Science and Technology, Yusung-Gu, Taejon, Korea.
authors.forename: D C
authors.initials: DC
authors.lastname: Lee
authors.forename: S G
authors.initials: SG
authors.lastname: Lee
authors.forename: S P
authors.initials: SP
authors.lastname: Hong
authors.forename: M H
authors.initials: MH
authors.lastname: Sung
authors.forename: H S
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics; D000596:Amino Acids / Q000096:biosynthesis*; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D010957:Plasmids; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids; D004800:Enzymes, Immobilized; D000581:Amidohydrolases; C020047:dihydropyrimidinase