Chromate-reducing activity of Hansenula polymorpha recombinant cells over-producing flavocytochrome b2

In spite of the great interest to studies of the biological roles of chromium, as well as the toxic influence of Cr(VI)-species on living organisms, the molecular mechanisms of chromate bioremediation remain vague. A reductive pathway resulting in formation of less toxic Cr(III)-species is suggested to be the most important among possible mechanisms for chromate biodetoxification. The yeast l-lactate:cytochrome c-oxidoreductase (flavocytochrome b2, FC b2) has absolute specificity for l-lactate, yet is non-selective with respect to its electron acceptor. These properties allow us to consider the enzyme as a potential candidate for chromate reduction by living cells in the presence of l-lactate.A recombinant strain of thermotolerant, methylotrophic yeast Hansenula polymorpha with sixfold increased FC b2 enzyme activity (up to 3 μmol min−1 mg−1 protein in cell-free extract) compared to the parental strain was used for approval our suggestion. The recombinant cells, stored in dried state, as well as living yeast cells were tested for chromate-reducing activity in vitro in the presence of l-lactate (as an electron donor for chromate reduction) and different low molecular weight, redox-active mediators facilitating electron transfer from the reduced form of the enzyme to chromate (as a final electron acceptor): dichlorophenolindophenol (DCPIP), Methylene blue, Meldola blue, and Nile blue. It was shown that the highest chromate-reducing activity of the cells was achieved in the presence of DCPIP.The ability of chromate to catch electrons from the reduced flavocytochrome b2 was confirmed using purified enzyme immobilized on the surface of a platinum electrode. The increasing concentration of Cr(VI) resulted in a decrease of enzyme-mediated current generated on the electrode during l-lactate oxidation. The shift and drop in amplitude of the peak in the cyclic voltammogram are indicative of Cr(VI)-dependent competition between reaction of chromate with reduced FC b2 and direct electron transfer from the enzyme to the electrode surface.The application of the chromate-reducing ability of FC b2-over-producing recombinant cells of H. polymorpha toward chromate bioremediation and the construction of cells-based biosensor for chromate monitoring in the environment are discussed.

Competitive effect of chromate on current generated during oxidation of l-lactate by flavocytochrome b2 (FC b2) immobilized on Pt electrode. Recombinant yeast cells over-producing FC b2 have an increased reductive bioremediation activity toward chromate.Figure optionsDownload as PowerPoint slideResearch highlights
► Ability of flavocytochrome b2 (FC b2) to reduce chromate has been demonstrated.
► Yeast cells over-producing FC b2 have an increased Cr(VI) bioremediation capacity.
► Redox mediator dichlorophenolindophenol facilitates chromate-reducing activity.