Clonagem do gene nahB de Pseudomonas putida G7 e expressão da enzima NahB envolvida na degradação do naftaleno, um componente do petróleo

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds consisting of two or more fused aromatic rings. Most of them are mutagenic and carcinogenic to humans and animals. PAHs are released through the environment mainly by anthropogenic activities related to the extraction, transport, refine, transformation and use of petroleum and its derivatives. Bioremediation is a strategy for PAHs elimination from the environment which uses enzymes or microorganisms displaying the capacity to metabolize these compounds and transform them into inert substances. Naphthalene is one of the most commonly found PAHs in the environment. The degradation of naphthalene by Pseudomonas sp. is an alternative procedure for the remediation of the environment with this pollutant. In Pseudomonas putida G7 the catabolic genes are organized in two operons on NAH7 plasmid: one encoding the upper-pathway enzymes involved in the conversion of naphthalene to salicylate, the second encoding the lower-pathway enzymes involved in the conversion of salicylate to tricarboxylic acid cycle intermediates. The nahB gene is responsible for codification of the protein cis-1,2-dihydro-1,2-dihydroxynaphthalene-1,2-dehydrogenase (NahB), which is the second enzyme from the upper-pathway. In the present work we present the cloning of nahB gene into the vector pCR®2.1-TOPO and the subcloning into the expression vectors pET-28a-GST-TEV and pET-28a-TEV. The heterologous expression was performed in RosettaTM (DE3) and ArticExpressTM (DE3). The expression vector pET-28a-GST-TEV-NahB, responsible for encoding the enzyme GST-NahB, was sequenced and the recombinant protein is correct. This was expressed in the soluble fraction at low temperatures in RosettaTM (DE3) at 20ºC and in ArticExpressTM (DE3) at 12ºC. The purification was performed by affinity chromatography and anion exchange chromatography. After purification, was conducted the cleavage with protease TEV. The protein NahB from the GST-NAHB remains with three amino acid residues than the native sequence and was called GGS-NAHB. The molecular mass of this recombinant enzyme was determined by mass spectrometry and the value was very close to theoretical. Structural characteristics of the enzyme GGS-NahB were measured by Dynamic Light Scattering and Circular Dichroism. By DLS, it was found that the protein is monodisperse and in tetrameric form in solution. CD experiments revealed an enzyme with approximately 25% -helix, 30% of parallel and antiparallel -sheet, 15% of beta turn and 40% of random coil. Further purification is being conducted for protein crystallization assays. The expression vector pET-28a-TEV-NahB, was also sequenced and the recombinant protein His-NahB presents, after cleavage with TEV protease, instead of two amino acid residues than the native sequence, 21 residues. The gene nahB will again subcloned into vector pET-28a-TEV for expression of His-NahB.