Direct Electrochemical Regeneration of Enzymatically Active 1,4- NADH Using a Nickel Modified Glassy Carbon Electrode

Amardeep Gill, Irshad Ali and Sasha OmanovicIntroduction

NADH is a cofactor used in many redox reactions. Among its uses in medical and biotechnological research, it is used to synthesize new high-value compounds such as pharmaceuticals, food additives, perfumes and pesticides.The reduction of NAD+ to 1,4-NADH occurs in two steps:

1st electron transfers2nd electron and H+ transfer

The second step produces two possible products: 1,4 NADH (active) NAD2 (inactive)

Problem: The kinetics of the formation of NAD2 are much faster due to the dimerization reaction than that of 1,4 NADH resulting in low yields of 1,4-NADH. Also, it is very expensive ($3050/mol). Regeneration would make 1,4-NADH much more economically available for industrial applications in which it has to be used.

Research Focus

1.Development and optimization of Ni Nano-island modified GCE

2.Determine the efficiency of the developed electrode to regenerate enzymatically active 1,4-NADH in a batch electrochemical reactor at different electrolysis potentials.

1mM: does not produce significant coverage 3mM : The Nano-islands are much bigger than desired but does produce

excellent coverage2mM: Produces Nano-Islands with uniform coverage and acceptable size

Techniques and Experimental Setup• Cyclic Voltammetry• Chrono Amperometry• UV-Vis Spectroscopy

Electrochemical CelloWorking electrode: Nickel modified GCE (Ni-GCE)oCounter Electrode: Graphite rodoReference Electrode: MSEoElectrolyte: 0.1M phosphate buffer solution pH 5.8 (Oxygen free)

Conclusions

The surface of glassy carbon was modified with Nickel Nano-particles resulting in good and uniform coverage. Optimum conditions for the modification were:

>2mM nickel nitrate in pH 4 acetate buffer solution

>10 scans of cyclic voltammetry from 0.0V to -1.8V at a scan rate of 50 mV/s.

The Ni Nano-islands served as a physical barrier which prevented the formation of NAD2 and increased the kinetics of the formation of 1,4 NADH by providing H+ right at the reaction site

The percentage of regenerated enzymatically active 1,4-NADH was found to be dependent on electrode potential

The highest yield: 97.4% of enzymatically active 1,4-NADH was regenerated at -1.5VMSE

In comparison with bare GCE, almost the same percentage of enzymatically active 1,4-NADH was regenerated but at a much lower overpotential (800mV more positive)

Enzymatic Assay showing 97.4% recovery of 1,4 active NADH. The following reaction is used to deplete 1,4 NADH in order to calculate the activity.

AcknowledgementsI would like to give a big thanks to:

-The S.U.R.E program and Prof. Omanovic for the opportunity-Irshad Ali for mentoring me through all the experiments -The rest of the members from the Electrochemistry lab for all their help through out the summer.

2mM nickel nitrate in pH 4 acetate buffer

Results

1mM nickel nitrate in pH 4 acetate buffer

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Monitoring the progress of the electrolysis by measuring the absorbance at 340 nm.

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