Expression and characterization of the TNT nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli.
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.
Lee
Bheong-Uk
BU
Kosin University, Busan, 606-701, Republic of Korea.
Park
Sung-Chul
SC
Cho
Yun-Seok
YS
Kahng
Hyung-Yeel
HY
Oh
Kye-Heon
KH
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D007700:Kinetics; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009243:NAD / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014303:Trinitrotoluene / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D009243:NAD; D014303:Trinitrotoluene; D009601:Nitroreductases