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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

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Journal of Industrial Microbiology & Biotechnology

Abstract

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.

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Acknowledgments

CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and Petrobras supported this work. We thank to Iuri Bastos M.Sc. (UFRJ) for the densitometry analysis using the QuantiScan 1.25 program, Dr. Márcio Schwaab and Prof. José Carlos Pinto (COPPE/UFRJ) for fruitful discussions, and the reserchers of the Setor de Imunobiológicos laboratory at INCQS/Fiocruz for making available the QuantiOne 4.4.1 program.

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Authors and Affiliations

  1. Programa de Engenharia Química-COPPE, 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 Leites Larentis, Haryana de Cássia Cunha Sampaio & Tito Lívio Moitinho Alves

  2. Instituto de Bioquímica Médica, Laboratório de Biologia Molecular, Universidade Federal do Rio de Janeiro–UFRJ, Centro de Ciências da Saúde (CCS), Bloco D, Cidade Universitária, Ilha do Fundão , Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil

    Ariane Leites Larentis, Haryana de Cássia Cunha Sampaio & Orlando Bonifácio Martins

  3. Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas–UNICAMP, Cidade Universitária Zeferino Vaz, Barão Geraldo, Campinas, SP, 13083-970, Brazil

    Maria Isabel Rodrigues

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  1. Ariane Leites Larentis
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Larentis, A.L., Sampaio, H.C.C., Martins, O.B. et al. 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. J Ind Microbiol Biotechnol 38, 1045–1054 (2011). https://doi.org/10.1007/s10295-010-0879-2

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