Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design.
Carbazole 1,9a-dioxygenase (CarA), the first enzyme in the carbazole degradation pathway used by Pseudomonas sp., was expressed in E. coli under different conditions defined by experimental design. This enzyme depends on the coexistence of three components containing [2Fe-2S] clusters: CarAa, CarAc, and CarAd. The catalytic site is present in CarAa. The genes corresponding to components of carbazole 1,9a-dioxygenase from P. stutzeri were cloned and expressed by salt induction in E. coli BL21-SI (a host that allows the enhancement of overexpressed proteins in the soluble fraction), using the vector pDEST™14. The expression of these proteins was performed under different induction conditions (cell concentration, temperature, and time), with the help of two-level factorial design. Cell concentration at induction (measured by absorbance at 600 nm) was tested at 0.5 and 0.8. After salt induction, expression was performed at 30 and 37°C, for 4 h and 24 h. Protein expression was evaluated by densitometry analysis. Expression of CarAa was enhanced by induction at a lower cell concentration and temperature and over a longer time, according to the analysis of the experimental design results. The results were validated at Abs (ind) = 0.3, 25°C, and 24 h, at which CarAa expression was three times higher than under the standard condition. The behavior of CarAc and CarAd was the inverse, with the best co-expression condition tested being the standard one (Abs (ind) = 0.5, T = 37°C, and t = 4 h). The functionality of the proteins expressed in E. coli was confirmed by the degradation of 20 ppm carbazole.
Laboratório de Bioprocessos, Universidade Federal do Rio de Janeiro-UFRJ, Centro de Tecnologia (CT), G115, Cidade Universitária, Ilha do Fundão, Caixa Postal 68502, Rio de Janeiro, RJ, 21945-970, Brazil. ariane@peq.coppe.ufrj.br
Ariane Leites
AL
Larentis
Haryana de Cássia Cunha
Hde C
Sampaio
Orlando Bonifácio
OB
Martins
Maria Isabel
MI
Rodrigues
Tito Lívio Moitinho
TL
Alves
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D049308:Dioxygenases; C108434:carbazole 1,9a-dioxygenase; D005472:Fluorouracil