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Applied Biochemistry and Biotechnology

, Volume 143, Issue 1, pp 1–15 | Cite as

Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

  • Brian L. Hassler
  • Megan Dennis
  • Maris Laivenieks
  • J. Gregory Zeikus
  • Robert M. WordenEmail author
Article

Abstract

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2° ADH). This mutation increased the K m(app) for NADP+ 200-fold while decreasing the K m(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7 × 104 times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.

Keywords

Secondary alcohol dehydrogenase Biosensor Cofactor regeneration Site-directed mutagenesis Bioelectronic Biocatalysis Electron mediator Toluidine blue NADP+ NAD+ 

Notes

Acknowledgments

The authors would like to thank the Michigan Technology Tri-Corridor (MTTC) program of the Michigan Economic Development Corporation (MEDC) and the IRGP programs at Michigan State University (MSU) for funding this work. Analytical support provided by the analytical chemistry facility in the MSU Department of Chemistry is also gratefully acknowledged.

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Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Brian L. Hassler
    • 1
  • Megan Dennis
    • 1
  • Maris Laivenieks
    • 2
  • J. Gregory Zeikus
    • 2
  • Robert M. Worden
    • 1
    Email author
  1. 1.Department of Chemical Engineering and Materials ScienceMichigan State UniversityEast LansingUSA
  2. 2.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA

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