[Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli].
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.
Hao
Shu-Feng
SF
Beijing Institute of Biotechnology, Beijing, Chinese Academy of Sciences, Shenyang, China.
Zhang
Wei-Cai
WC
Li
Ying-Li
YL
Yuan
Hong-Jie
HJ
Huang
Liu-Yu
LY
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
D006827:Hydantoins; C031427:5-benzylhydantoin; D010649:Phenylalanine; D000581:Amidohydrolases; C451299:N-carbamoylamino acid amidohydrolase