[Cloming, sequence analysis of imidase gene from Alcaligenes eatrophus and its expression in E. coli].
A hydantoin-cleaving microorganism 112R4 is screened and identified to be Alcaligenes eutrophus. The resting cell of Alcaligenes eutrophus 112R4 can catalyze the hydrolysis of hydantoin, dihydropyrimidine and succinimide effectively, but not function to 5-monosubstituted hydantoins or 5,5'-disubstituted hydantoins. The microorganism can utilize succinimide as a sole carbon source and nitrogen source, which indicates the presence of a complete transformation pathway of succinimide, and a hydantoin-cleaving enzyme, imidase, is suggested to be contained in this metabolic pathway. A 6 kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R4 is shown to be correlative with the transformation of succinimide. A 2 kb DNA fragment containing the gene of imidase is subcloned and sequenced. Deletion analysis verifies that one open reading frame of 876 nucleotides, which encodes a peptide of 291 amino acids, with a calculated molecular weight of 33688, is responsible for the encoding of imidase. This is the first report of the nucleotide and amino acid sequences of imidase (GenBank accession number: AF373287). A homology search performed in protein database reveals an identity of 14% with polysaccharide deacetylase conserved domain, an identity of 60% with N-terminal 20 amino acids of Blastobacter sp. A17p-4, but no apparent similarity with all known cyclic-amide-cleaving enzymes. This result suggested that the imidase should be classified as a new member of cyclic amidases. Under the control of lac promoter and IPTG induction, the imidase activity of transformed E. coli reached 3200 U/L, which is about 7-fold higher than that of gene donor strain.
Wang
Yu
Y
Institute of Microbiology, The Chinese Academy of Science, Beijing 100080, China.
Zhang
Yingzi
Y
Ding
Jiuyuan
J
Liu
Yangjian
Y
Wang
Jiang
J
Yu
Zhihua
Z
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000581:Amidohydrolases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016415:Sequence Alignment
D004269:DNA, Bacterial; D000581:Amidohydrolases; C020047:dihydropyrimidinase