Cloming, Sequence Analysis of Imidase Gene from Alcaligenes eatrophus and Its Expression in E. coli
真养产碱杆菌112R4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达

A hydantoin-cleaving microorganism 112R 4 is screened and identified to be Alcaligenes eutrophus. The resting cell of Alcaligenes eutrophus 112R 4 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 6kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R 4 is shown to be correlative with the transformation of succinimide. A 2kb 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 3200U/L, which is about 7-fold higher than that of gene donor strain.