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

Area of Science:

• Biochemistry
• Enzymology
• Biosensor technology

Background:

• The alpha-hydroxy acid oxidoreductase (HAOx) family includes enzymes crucial for L-lactate detection in biosensors.
• Current L-lactate biosensor development is limited by the scarcity and lack of diversity in available L-lactate-oxidizing enzymes, primarily relying on Aerococcus viridans L-lactate oxidase (AvLOx).

Purpose of the Study:

• To identify and characterize novel L-lactate oxidases (LOx) as alternatives to the commercially available AvLOx.
• To evaluate the suitability of these newly discovered enzymes for integration into L-lactate biosensor configurations.
• To investigate the relationship between biochemical and electrochemical performance of L-lactate oxidases in biosensor applications.

Main Methods:

• Discovery and characterization of novel L-lactate oxidases from the HAOx family.
• Expression of FMN-dependent L-lactate oxidases in E. coli for potential large-scale production.
• Electrochemical characterization of enzyme performance using a mediated biosensor setup.

Main Results:

• Identification of seven novel L-lactate oxidases with narrow substrate specificity and varied kinetic efficiencies for L-lactate.
• Successful expression of some enzymes in E. coli, indicating potential for commercial production.
• Electrochemical performance of the new enzymes was comparable or superior to the commercial AvLOx.
• Demonstration that electrochemical performance does not directly correlate with biochemical performance, complicating enzyme selection for biosensors.

Conclusions:

• The discovery of new L-lactate oxidases significantly expands the enzyme toolbox for L-lactate biosensor development.
• These novel enzymes offer promising alternatives to AvLOx, potentially leading to improved L-lactate detection.
• Further research is needed to understand the complex relationship between enzyme kinetics and biosensor performance for accurate enzyme selection.