title: 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 (k(cat)/K(M)= 1.1×10(5) M(-1) s(-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.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Robins
authors.forename: David O
authors.initials: DO
authors.lastname: Hooks
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D001388:Aziridines; D001673:Biodegradation, Environmental; D002274:Carcinogens, Environmental / Q000378:metabolism*; D002474:Cell-Free System; D002857:Chromium / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004800:Enzymes, Immobilized / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D008863:Microspheres; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics
chemical_list: D001388:Aziridines; D002274:Carcinogens, Environmental; D017931:DNA Primers; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D002857:Chromium; C074702:chromium hexavalent ion; C100099:tretazicar; D010088:Oxidoreductases; C063243:chromate reductase