title: [Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli].
abstract: Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E. coli. The gene was highly expressed in E. coli M15 under control of T5 promoter. A protein band about 44kD was detected by SDS-PAGE in the recombinant cell lysate. The objective product, which is principally in soluble form, represented 40% of total cell protein. The N-carbamoylase specific activity of the recombinant M15/pQE60- hyuC is 53 times higher than that of Arthrobacter BT801. The total biotransformation activity increased 8.1 times when. M15/pQE60-hyuC was added into the Arthrobacter BT801 reaction system. The successful expression of the enzyme is significant for the application of the hydantoinase producing strain or the enzyme thereof.
authors.affiliation: Beijing Institute of Biotechnology, Beijing, Chinese Academy of Sciences, Shenyang, China.
authors.forename: Shu-Feng
authors.initials: SF
authors.lastname: Hao
authors.forename: Wei-Cai
authors.initials: WC
authors.lastname: Zhang
authors.forename: Ying-Li
authors.initials: YL
authors.lastname: Li
authors.forename: Hong-Jie
authors.initials: HJ
authors.lastname: Yuan
authors.forename: Liu-Yu
authors.initials: LY
authors.lastname: Huang
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006827:Hydantoins / Q000378:metabolism; D008957:Models, Genetic; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D006827:Hydantoins; C031427:5-benzylhydantoin; D010649:Phenylalanine; D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase