More Than One Enzyme: Exploring Alternative FMN-Dependent L-Lactate Oxidases for Biosensor Development

Abstract

The α-hydroxy acid oxidoreductase (HAOx) family contains a diverse group of enzymes that can be applied in biosensors for L-lactate detection, most prominently lactate oxidase (LOx). The limited availability and a lack of diversity of L-lactate-oxidizing enzymes have currently hindered advancements in L-lactate biosensor development. Until now, the field has mostly relied on a single, commercially available enzyme, namely Aerococcus viridans L-lactate oxidase (AvLOx). In this study, we present newly discovered alternative L-lactate oxidases that exhibit a narrow substrate specificity and varied kinetic efficiencies toward L-lactate, making them suitable for integration into existing biosensor configurations. Some of these FMN-dependent L-lactate oxidases could be obtained in substantial amounts from routine E. coli expression, potentially facilitating commercial production. Using electrochemical characterization with a mediated biosensor setup, we present 7 enzymes that perform comparable or even better than commercial AvLOx. Finally, we show that their electrochemical performance is not directly correlating with their biochemical performance, making predictions of the suitability of enzymes for biosensor applications extremely difficult. Our research emphasizes the significance of expanding the enzyme toolbox of L-lactate oxidases for the development of improved L-lactate biosensors.

Figure 1

Reaction mechanism of L-lactate utilized in a biosensor in the presence of oxygen (O₂) as an electron acceptor and PB as a mediator immobilized on a working electrode (WE)^.

Figure 2

(A) Maximum likelihood phylogenetic tree of the 12 predicted LOx sequences expressed in this study. Sequences that were previously screened for substrate specificity are indicated with white labels, and sequences that are characterized electrochemically during this study are displayed within black rectangles. (B) AvLOx crystal structure with highlighted lid-loop region (blue) and FMN cofactor (magenta).

Figure 3

Sensor responses to L-lactate plotted against the amount of immobilized enzyme (assuming 100% efficiency of immobilization) on the sensor. Sensor responses are taken from the linear slopes of KL intercepts plotted versus their corresponding L-lactate concentrations of 50–250 μM (n = 3).

Figure 4

Comparison of apparent rate constants for LOxs immobilized on RDEs in a PEDOT:PSS–PB matrix (k₂) with the respective apparent rate constants measured in solution (k_cat). k₂ values were estimated from the Koutecký–Levich analysis and describe the overall enzyme reaction rate on the sensor.