Anisotropic Oxidative Growth Of Goethite-Coated Sand Particles In Column Reactors During 4-Chloronitrobenzene Reduction by Fe(II)/Goethite

Reduction of nitroaromatic compounds (NACs), an important class of groundwater pollutants, by Fe(II) associated with iron oxides, a highly reactive reductant in anoxic aquifers, has been studied widely, but there...

A low-dimensional N-rich coordination polymer as effective fluorescence sensor for 2,4,6-trinitrophenol detection in an aqueous medium

2,4,6-Trinitrophenol (TNP) is highly explosive and toxic in nitroaromatic compounds. The selective detection of TNP is crucial for life safety and the social environment. As a novel multifunctional material, coordination...

A Complementary Silicon Quantum Dot-Enzyme Platform for Selective Detection of Nitroaromatics Compounds

To address the issue of poor selectivity in nanotechnology-driven, portable nitroaromatics sensors, we have coupled a ratiometric photoluminescence sensor based on silicon quantum dots and fluorescent proteins with a colorimetric enzyme-based sensor. Together, the sensors allow differentiation of nitroaromatic compounds – specifically, distinguishing acetylcholinergic nerve agents from the explosive compounds explored herein. The combined system can detect 2,4,6-trinitrotoluene, 2,4-dinitrotoluene and 4-nitrophenol with micromolar detection limits and affords subsequent differentiation from the nitro-containing nerve agent paraoxon. This demonstrates the advantage of merging elements of materials chemistry and biochemistry to devise customized sensors which can accurately identify hazardous chemical species.

A Complementary Silicon Quantum Dot-Enzyme Platform for Selective Detection of Nitroaromatics Compounds

To address the issue of poor selectivity in nanotechnology-driven, portable nitroaromatics sensors, we have coupled a ratiometric fluorescence sensor based on silicon quantum dots and fluorescent proteins with a colorimetric, enzyme-based sensor. Together, the sensors allow differentiation of nitroaromatic compounds – specifically, distinguishing acetylcholinergic nerve agents from the explosive compounds explored herein. The combined system can detect 2,4,6-trinitrotoluene, 2,4- dinitrotoluene and 4-nitrophenol with micromolar detection limits and affords subsequent differentiation from the nitro-containing nerve agent paraoxon. This demonstrates the advantage of merging elements of materials chemistry and biochemistry to devise customized sensors which can accurately identify hazardous chemical species.

Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish. There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels. The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish. There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels. The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

Electrochemical Study of Butyl-Pyrene Nitrobenzoate Derivatives Trapped on MWCNT Nanostructured Electrodes

In the scope of our studies tending to find new nanostructured electrodic platforms containing nitroaromatic compounds (NACs) capable of generating in situ electrocatalytic redox couples, we synthesized and electrochemically studied three related 4-(pyren-1-yl)-butyl-substituted nitrobenzoates (2-NBPy, 3-NBPy and 4-NBPy). The design of the compounds is based on a combination of a) an adsorptive tail (-butyl-pyrene) capable of interacting via π-π stacking with the MWCNT nanostructured electrodes and b) nitroaromatic compounds (NACs) capable of electrochemically activating to form a RNHOH/NO redox couple trapped on the nanostructured electrodic platform. Morphological and structural analyses of the nanostructured interfaces were performed by SEM and WAXS/SAXS analysis. All of the NBPy compounds trapped on the nanostructured electrodic platform were susceptible to reduction, generating the corresponding hydroxylamine derivative. The order of ease of reduction for the nitrocompounds is 4-NBPy > 2-NBPy > 3-NBPy. After electrochemical activation, all compounds generated an RNHOH/NO redox mediator couple with the following order of stability of the mediator couple: 2-NBPy > 3-NBPy > 4-NBPy. For the 2-NBPy and 3-NBPy derivatives, excellent stability of the couple was observed, and a decrease in the peak current of 6% was observed after 60 minutes.