Escherichia coli NemA Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium

Abstract

Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI) to insoluble and relatively non-toxic Cr(III), bacterial bioremediation of Cr(VI) pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI) remediation. To identify novel Cr(VI) reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI) indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI) reductase (kcat/KM = 1.1×105 M−1s−1 with NADH as cofactor). Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI) remediation.

Plasmidic Expression ofnemAandyafC* Increased Resistance of Ethanologenic Escherichia coli LY180 to Nonvolatile Side Products from Dilute Acid Treatment of Sugarcane Bagasse and Artificial Hydrolysate

“…NemA is a versatile NADPH-dependent flavoprotein reductase (old yellow enzyme) capable of reducing a broad range of organic compounds, including electrophiles (quinones, glyoxals, trinitrotoluene) (27) and even inorganic substrates, such as nitrates and chromates (28,34). Considering the diversity of compounds formed by acid treatment of xylose (17,35), it is not surprising that some components of PXV (Fig.…”

“…NemA reductase activity was measured as the N-ethylmaleimide (NEM)-dependent oxidation of NADPH (27,28) unless stated otherwise. For enzyme assays, cells (50 ml) grown in AM1 xylose broth containing 100 mM morpholinepropanesulfonic acid (MOPS) (pH 7) were harvested by centrifugation at an OD 550 of approximately 1, washed twice with 10 ml of cold potassium phosphate buffer (50 mM; pH 7.0), and resuspended in phosphate buffer (3 ml).…”

Recombinant surface engineering to enhance and expand the potential of biologically produced nanoparticles: A review

“…The second report dealt with hexavalent chromium (Cr(VI)) remediation. A Cr(VI) reductase, NemA, was fused to PhaC R.eu for presentation on the granule surface [66]. The study demonstrated that the NemAPhaC enzyme combination-displaying granules together with NADH cofactor-generating enzymes such as glucose dehydrogenase or formate dehydrogenase enabled a high degree of chromium transformation with less depletion of NADH cofactors.…”

Hazards, Risks, and Low Hazard Development Paths of Synthetic Biology

“…An example for an application which abandons the full spectrum of functionalities of living organisms and concentrates only on relevant mechanisms is the application of chromate reductase coupled on polyhydroxyalkanoate granules, developed by Robins and colleagues. In combination with a cofactor regenerating enzyme 25 this quite "plain" system for ex situ remediation can transform the toxic and unfortunately water soluble industrial waste product hexavalent chromium into a nontoxic state (Robins et al 2013). Systems based on this combination were suggested as an economical and safe solution for remediation of a number of pollutants (e.g.…”