Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli.
- Author:
Shu-Feng HAO
1
;
Wei-Cai ZHANG
;
Ying-Li LI
;
Hong-Jie YUAN
;
Liu-Yu HUANG
Author Information
1. Beijing Institute of Biotechnology, Beijing, Chinese Academy of Sciences, Shenyang, China.
- Publication Type:Journal Article
- MeSH:
Amidohydrolases;
genetics;
metabolism;
Arthrobacter;
enzymology;
genetics;
Electrophoresis, Polyacrylamide Gel;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
genetics;
Hydantoins;
metabolism;
Models, Genetic;
Phenylalanine;
metabolism;
Plasmids;
genetics;
Polymerase Chain Reaction
- From:
Chinese Journal of Biotechnology
2003;19(2):174-177
- CountryChina
- Language:Chinese
-
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.
