1. Semnan University 2. Semnan University, Semnan, Iran 3. Dep. of Biotechnology, Semnan University, Semnan, Iran
Antibiotic- resistant pseudomonas aeruginosa play an important role in the pathogenesis of human diseases. bioinformatic's studies indicate dimethylaniline monooxygenase exist in pseudomonas aeruginosa. it is able to metabolize drugs and some antibiotics, such as imipenem as well as the human fmo-dependent enzymes.
In this study, dimethylaniline monooxygenase was studied in silico. however, practically the gene from pseudomonas aeruginosa was amplified by pcr, cloned in a pet-22b and expressed heterologously in e.coli bl21 (de3). the recombinant proteins were analyzed by sds-page method and purified by affinity chromatography with his-tag residues.
The results revealed that the enzyme was >99 % identical to dimethylaniline monooxygenase within pseudomonas species, and among the strains of p. aeruginosa. the gene was 1353 bp in length, which expressed a 450-amino acid, 49 kda protein. the expression of the gene was induced by 1mm iptg. the sds/page gel analysis showed the protein in 47 kda molecular weight. functional analysis of the isolated dimethylaniline monooxygenase is under processing.
These findings open new perspectives in human oxidative metabolism.