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Functional Expression of a Mo-Cu-Dependent Carbon Monoxide Dehydrogenase (CODH) and Its Use as a Dissolved CO Bio-microsensor

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  • Stacy Simai Reginald, Gwangju Institute of Science and Technology
  • ,
  • Michael Etzerodt
  • ,
  • Deby Fapyane
  • ,
  • In Seop Chang, Gwangju Institute of Science and Technology

Herein, we report the heterologous expression in Escherichia coli of a Mo-Cu-containing carbon monoxide dehydrogenase (Mo-Cu CODH) from Hydrogenophaga pseudoflava, which resulted in an active protein catalyzing CO oxidation to CO2. By supplying the E. coli growth medium with Na2MoO4 (Mo) and CuSO4 (Cu), the Mo-Cu CODH metal cofactors precursors, the expressed L-subunit was found to have CO-oxidation activity even without the M- and S- subunits. This successful expression of CO-oxidizing-capable single L-subunit provides direct evidence of its role as the catalytic center of Mo-Cu CODH that has not been discovered and studied before. Subsequently, we used the expressed protein to construct a CO bio-microsensor based on a newly developed fast and sensitive Clark-type CO2 transducer using an aprotic solvent/ionic liquid electrolyte. The CO bio-microsensor exhibited a linear response to CO concentration in the 0-9 μM range, with a limit of detection (LOD) of 15 nM CO. The sensor uses a mixture of Mo-Cu CODH's L-subunit/Mo, Cu cofactors/methylene blue, confined in the enzyme chamber that is placed in front of a CO2 transducer. The optimized sensor's sensitivity and performance were retained to levels of at least 80% for 1 week of continuous polarization and operation in an aqueous medium. We have also demonstrated the use of an alkaline front-trap solution to make a completely O2/CO2 interference-free microsensor. The CO bio-microsensor developed in this study is potentially useful as an analytical tool for the detection of trace CO in dissolved form for monitoring dissolved CO concentration dynamics in natural or synthetic systems.

TidsskriftACS Sensors
Sider (fra-til)2772–2782
StatusUdgivet - jul. 2021

Bibliografisk note

Funding Information:
We thank Niels Peter Revsbech for the guidance in sensor design and construction. This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean Government (2020R1A2C3009210) and the C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2021M3D3A1A01079730). BK21 Plus supported a research stay at Aarhus University, Denmark. We also thank the Grundfos Foundation for its support to the sensor projects at Aarhus University Centre of Water Technology (WATEC). Technical support by Anette Kjems, Lars Borregaard Pedersen, and Mette L-G. Nikolajsen is gratefully acknowledged.

Publisher Copyright:
© 2021 American Chemical Society.

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