Title

Studying TNT reduction in E. coli: Pathway and kinetic approaches

Date of Completion

January 2006

Keywords

Engineering, Environmental

Degree

Ph.D.

Abstract

This research aimed to elucidate the mechanism and kinetics of the microbiological reductive transformation of TNT. Escherichia coli was used as a model microorganism because of pre-existing biochemical knowledge on its nitroreductases. The pathway and kinetics of TNT reductive transformation were thoroughly studied and kinetic models were proposed for TNT transformation processes with the whole cells of Escherichia coli. The roles of oxygen-insensitive nitroreductases NfsA and NfsB on the TNT reductuctive transformation pathway were studied with partially complementary strain Escherichia coli JVQ2 (pTAA) (nfsA+nfsB -), E. coli JVQ2 (pJAB) (nfsA -nfsB+) and double mutant strain E. coli JVQ2 (nfsA-nfsB-). Efforts were taken to control the TNT transformation pathway to certain stage (hopefully 2ADNT and 4ADNT) with the cell extracts of E. coli JVQ2 (pTAA). However, TNT transformation did not proceed beyond the stage of HADNT, suggesting other enzymes are involved in the reduction of TNT to the level of ADNT and/or DANT. ^ The role of NfsA and NfsB in TNT reduction was studied using the double mutant E. coli JVQ2 (nfsA-nfsB -) and the genetically complemented strains E. coli JVQ2 (pTAA) (nfsA+nfsB-) and E. coli JVQ2 (pJAB) (nfsA-nfsB +). Their activities towards the three substrates varied significantly. With similar expression level of NfsA and NfsB, E. coli JVQ2 (pTAA) transformed TNT almost 8-fold faster than strain E. coli JVQ2 (pJAB), and 26-fold faster than strain JVQ2. In addition, the nfsA+ strain exhibited a higher activity against 2A46DNT (0.098 μmole•mg protein-1•min -1) than the nfsB+ strain, but both had low activity against 4A26DNT (4 ∼ 5 nmole•mg protein-1 •min-1). Negligible reduction occurred in the ortho-position during the NfsA-mediated TNT transformation, in contrast to high production in the NfsB-containing strain. The double mutant strain E. coli JVQ2 retained very low nitroreductase activity. Hence, all the three nitroreductases, NfsA, NfsB and putative nitroreductase NfsC are able to catalyze TNT reduction, with NfsA displaying the highest activity. ^ TNT reduction catalyzed by NfsA was studied using the cell extacts of E. coli JVQ2 (pTAA). Surprisingly, TNT was stoichiometrically converted to HADNT, but no reduction beyond HADNT was detected. Enzyme inactivation was observed. Transformation capacity (Tc), estimated at 54 to 395 μmol TNT/mg NfsA, increased with initial TNT conditions. A simplified Ping Pong Bi Bi kinetic expression incorporating the transformation capacity term achieved favorable best-fits to the complete TNT reductive profiles, revealing a strong dependence on TNT, but not NADPH. (Abstract shortened by UMI.)^