Electrochemical characterization of a dual cytochrome-containing lactate dehydrogenase

Anastasya Boyarski, Noam Shlush, Shiraz Paz, Jerry Eichler, Lital Alfonta

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Flavin-dependent L-lactate dehydrogenase (LDH) from baker's yeast (Saccharomyces cerevisiae) reversibly catalyzes the oxidation of L-lactate to L-pyruvate. In this study, four different enzymatic constructs were generated, and their catalytic and electrochemical properties were compared. Specifically, a truncated form of the native enzyme that includes only the catalytic domain, the native enzyme that includes an intrinsic electron-transferring cytochrome b2, a novel artificial enzyme containing a minimal cytochrome c and a version of the enzyme containing a fusion between two cytochromes were designed. All four variants were successfully expressed in Escherichia coli and presented properly matured heme domains. Assessing in vitro biocatalytic performance as reflected by lactate oxidation revealed the fusion-containing enzyme to be ∼ 12 times more active than the native enzyme. Electrochemical studies of electrode drop-casted enzyme variants also showed the superior performance of the dual-cytochrome construct, which displayed a lower average redox-potential for lactate oxidation, oxygen insensitivity in the lactate oxidation potential range and a wider dynamic range for lactate sensing, relative to the native enzyme. Moreover, product inhibition of this variant occurred at much higher lactate concentrations than with the native enzyme. In addition, when lower potentials were scanned using cyclic voltammetry, lactate-dependent oxygen reduction was measured for the dual-cytochrome fusion enzyme.

Original languageEnglish
Article number108406
JournalBioelectrochemistry
Volume152
DOIs
StatePublished - 1 Aug 2023

Keywords

  • Flavin dependent lactate dehydrogenase
  • Lactate bio sensing
  • Lactate dehydrogenase engineering
  • Minimal cytochrome

ASJC Scopus subject areas

  • Biophysics
  • Electrochemistry
  • Physical and Theoretical Chemistry

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