Cloning and expression of meta-cleavage enzyme (CarB) of carbazole degradation pathway from Pseudomonas stutzeri

Larentis, Ariane Leites; Alves, Tito Lívio Moitinho; Martins, Orlando B.

doi:10.1590/s1516-89132005000400016

Cited by 3 publications

(2 citation statements)

References 13 publications

(17 reference statements)

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“…1): one containing the carAa gene and the other with carAc and carAd genes, besides the ORF7 gene, whose encoded protein is not essential to dioxygenase activity. The cloning procedures are described in detail in Larentis et al [25,26] for CarB (2 0 -aminobiphenyl-2,3-diol-1,2-dioxygenase), the second enzyme of the P. stutzeri carbazole degradation pathway (Fig. 1).…”

Section: Strain and Plasmidmentioning

confidence: 99%

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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

Larentis

Sampaio

Martins

et al. 2010

J Ind Microbiol Biotechnol

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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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Section: Strain and Plasmidmentioning

confidence: 99%

“…E. coli BL21-SI without any plasmid was used as a negative control. Cells with an empty plasmid were not used as control because the pDEST TM 14 vector contains a selection marker (ccdB gene), which inhibits growth of E. coli [26].…”

Section: Expressionmentioning

confidence: 99%

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

Larentis

Sampaio

Martins

et al. 2010

J Ind Microbiol Biotechnol

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A new halotolerant xylanase from Aspergillus clavatus expressed in Escherichia coli with catalytic efficiency improved by site-directed mutagenesis

Pasin,

Lucas,

de Oliveira

et al. 2024

3 Biotech

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Expression and Homology Modeling of 2;-Aminobiphenyl-2,3-Diol-1,2-Dioxygenase From Pseudomonas stutzeri Carbazole Degradation Pathway

Larentis

Almeida

Rössle

et al. 2006

CBB

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The enzyme 2'-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an alpha(2)beta(2) heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1,082-base pair polymerase chain reaction product corresponding to carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The alpha(2)beta(2)-heterotetrameric 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.

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Cloning and expression of meta-cleavage enzyme (CarB) of carbazole degradation pathway from Pseudomonas stutzeri

Cited by 3 publications

References 13 publications

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

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

A new halotolerant xylanase from Aspergillus clavatus expressed in Escherichia coli with catalytic efficiency improved by site-directed mutagenesis

Expression and Homology Modeling of 2;-Aminobiphenyl-2,3-Diol-1,2-Dioxygenase From Pseudomonas stutzeri Carbazole Degradation Pathway

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