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Chromate reductase (codon-optimized for C. reinhardtii)

Chromate reductase (ChrR) is an oxidoreductase enzyme that is able to transfer 3 electrons towards Cr6+ anions, which results in reduction directly into Cr3+ ions. This reduction avoids production of side products such as Cr5+ and Cr4+, which are quite toxic to the cell. Both NADH and NADPH are equally utilized as electron donors. Other substrates that can be reduced: quinones (primary biological role), potassium ferricyanide, 2,6-dichloroindophenol, V5+, Mo6+, methylene blue, cytochrome c and U6+.

Reaction of electron transfer is the following:

2 NAD(P)H + Cr6+ + O2 = 2 NAD(P)+ + Cr3+ + superoxide

Flavin mononucleotide (FMN) and Ca2+ serve as cofactor molecules.

This enzyme lacks any post-translational modifications.

Optimal Conditions:

Optimum pH (for Cr6+) = 5

Optimum temperature (for Cr6+) = 35 degrees Celsius

Validation

Note: We struggled to optimize the protocol for Chromate reduction assay for about 3 months by changing the mass of variables and conditions, such as filtration, a method of protein extraction, an addition of protease inhibitors, keeping strict low-temperature conditions, pH adjustments and so on. The final results were obtained in the very last days.

Chromate reduction assay performed by use of two strains of algae C.reinhardtii, namely with cell wall (cw+) and without (cw-). The measurement of Cr(VI) concentration as a period of time was conducted according to 1,5-DPC method described in Chromate reduction assay protocol. Non-electroporated algae samples (without Chromate Reductase) and electroporated algae (with Chromium reductase) were used. NADH substrate for chromium reductase was tested as well.

As it can be seen from the graphs 1 and 2, the natural ability of ability of C.reinhardtii to reduce Cr(VI) to Cr(III) was observed. However, there is a noticeable increase in reduced ability of electroporated samples of cw+ strains (almost two-fold decrease in hexavalent chromium) due to the presence of the recombinant protein - chromium reductase.

Considering the reduction of hexavalent chromium in the electroporated cw- stains from graphs 3 and 4, it is possible to say that while in graph 3 there was approximately 20% decrease in Cr (VI) concentration, in the graph 4 - about 12%. It could be proposed that the addition of NADH influenced the reduction ability of the cw- strain. Reduction ability of electroporated CW- strains upon the addition of NADH was not as dramatic as in the electroporated cw+ strains. The natural ability of cw- strains to reduce Cr (VI) upon the addition of NADH constituted much higher reduction as compared to the NADH- control. It could be suggested that this happened due the influence of the NADH in the mutant strain of C.reinhardtii.

In the graph 3, it can be observed that experiment with CW- (NADH+) was done improperly due to human error, because reduction ability of electroporated and non-electroporated cells are almost the same.

Overall, according to the results of the experiment, it was observed that the presence of Chromate Reductase substantially improved the efficiency of hexavalent chromium reduction for C. reinhardtii.

We have experimentally proven that the part central to our project works as expected.

Sequence and Features