Bioengineered Lactate Oxidase Mutants for Enhanced Electrochemical Performance at Acidic pH

Abstract

Electrochemical lactate biosensors based on lactate oxidase (LOx) are used for diagnostics, sports medicine, and the food industry. However, samples from these sectors may have acidic levels at which the biocatalytic activity of LOx may be diminished. In this work, the enhancement of the bio‐electrocatalytic activity of LOx at low pH by pKa modulation of its catalytic His265 was studied by rational engineering of lactate oxidase from Aerococcus viridans (AvLOx). Several candidates based on interactions with His265 were selected by in silico structural analysis. The designed variants were heterologously produced, and the S175A mutant showed considerable improvement over its wild‐type counterpart, showing 157 % of the enzymatic activity found in the wild‐type at pH 5. Bioelectrodes were assembled based on Prussian‐blue‐modified carbon paper mediator system. The electrocatalytic performance of the amperometric biosensors of S175A variant at pH 5 exhibited a linear range of 0.2–2 mM measured at 0 V vs SCE, a sensitivity of −17.52 μA/mM ⋅ cm2, representing 240 % of the sensitivity found in the wild‐type biosensor, and a limit of detection of 38 μM, lower than observed with the wild‐type enzyme. These results show that the mutant obtained offers a significant improvement in lactate biosensing at low pH.