Gene cloning, expression, and substrate specificity of an imidase from the strain Pseudomonas putida YZ-26.
A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with N-terminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni(2+)-NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated.
Shi
Ya-wei
YW
Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of the National Ministry of Education, Shanxi University, 92 Wucheng Road, Taiyuan, Shanxi 030006, PRC. yaweishi@sxu.edu.cn
Cui
Li-fang
LF
Yuan
Jing-ming
JM
D000421:Alcaligenes / Q000235:genetics; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D041841:Bradyrhizobiaceae / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D021122:Protein Subunits; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000581:Amidohydrolases; C020047:dihydropyrimidinase