Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli-T7 expression system.
Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.
Bigey
F
F
Ecole Nationale Supérieure Agronomique-Institut National de la Recherche Agronomique, UFR de Microbiologie Industrielle et de Génétique des Microorganismes, Montpellier, France. bigey@ensam.inra.fr
Chebrou
H
H
Fournand
D
D
Arnaud
A
A
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics*; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D000581:Amidohydrolases; C085012:adipamidase; D006836:Hydro-Lyases; C048831:nitrile hydratase