Expression and Characterization of the TNT Nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli

Abstract

Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V (max) and K ( m ) values of the enzyme for TNT were 12.6 micromol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.

A kinetic analysis of three modified novel nitroreductases

“…The ability of bacterial enzymes to reduce nitroaromatic compounds is widespread and NTR enzymes catalysing the reduction of explosive compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and nitroglycerine (NG) have been identified (Caballero et al 2005;Lee et al 2008;Gonzalez-Perez et al 2007;Seth-Smith et al 2002;Blehert et al 1999). Reduction of TNT by Pseudomonas sp.…”

“…A second NTR form from JLR11 PnrB, showed negligible activity towards TNT in comparison with the PnrA. Lee et al (2008) isolated and characterised a PnrB homologue (93% identity) from Pseudomonas sp. strain HK-6, a strain able to utilise TNT and RDX as sole nitrogen sources.…”

Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

“…C1 [13], Phanerochaete chrysosporium [14], Pseudomonas sp. [15][16][17] and Actinomycetes [18] have been identified for their tolerance and activity to TNT. However, microorganisms do not possess sufficient biomass due to non-optimal growth conditions or low growth rate at contaminated sites, thus the degradation rate of TNT by microorganisms is limited [4].…”

Structure of the apo anti-influenza CH65 Fab

“…The recent explosion in the identification and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses has bolstered enthusiasm and efforts towards the design of universal flu vaccines and novel therapeutics. These bnAbs bind the influenza hemagglutinin glycoprotein (HA; Wilson et al, 1981) and target functionally conserved epitopes on the HA head and stem regions (Lee & Wilson, 2015). In conjunction with these advances in antibody discovery, the developmental pathways that the HA stem-targeted V H 1-69encoded antibodies take to convert from germline to affinitymatured forms are an active area of investigation (Lingwood et al, 2012;Avnir et al, 2014;Pappas et al, 2014).…”