Minimizing the effects of oxygen interference on L-lactate sensors by a single amino acid mutation in Aerococcus viridans L-lactate oxidase

Kentaro Hiraka, Katsuhiro Kojima, Chi En Lin, Wakako Tsugawa, Ryutaro Asano, Jeffrey LaBelle, Koji Sode

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

L-lactate biosensors employing L-lactate oxidase (LOx) have been developed mainly to measure L-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some L-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of L-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5 mM L-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9–12% due to oxygen interference. The Ala96Leu mutant maintained 56–69% of the response current at the same L-lactate level and minimized the relative bias error to −19% from −49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of L-lactate concentrations.

Original languageEnglish (US)
Pages (from-to)163-170
Number of pages8
JournalBiosensors and Bioelectronics
Volume103
DOIs
StatePublished - Apr 30 2018

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Keywords

  • Biomedical engineering
  • L-lactate
  • L-lactate oxidase
  • Oxygen
  • Screen-printed carbon electrode
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry

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