Wayne M. Patrick

l ‐Malate is a key flavor enhancer and acidulant in the food and beverage industry, particularly winemaking. Enzyme‐based amperometric biosensors offer convenience for monitoring its concentration. However, only a small number of off‐the‐shelf malate‐oxidizing enzymes have been used in previous devices. These typically have linear ranges poorly suited for the l ‐malate concentrations found in fruit processing and winemaking, making it necessary to use precisely diluted samples. Here, we describe a pipeline of database‐mining, gene synthesis, recombinant expression, and spectrophotometric assays to characterize previously untested enzymes for their suitability in biosensors. The pipeline yielded a bespoke biocatalyst—the Ascaris suum malic enzyme carrying mutation R181Q [AsME(R181Q)]. Our first prototype with AsME(R181Q) had an ultra‐wide linear range of 50–200 mM l ‐malate, corresponding to concentrations found in undiluted fruit juices (including grape). Changing the dication from Mg 2+ to Mn 2+ increased sensitivity five‐fold and adding citrate (100 mM) increased it another six‐fold, albeit decreasing the linear range to 1–10 mM. To our knowledge, this is the first time an l ‐malate biosensor with a tuneable combination of sensitivity and linear range has been described. The sensor response was also tested in the presence of various molecules abundant in juices and wines, with ascorbate shown to be a potent interferent. Interference was mitigated by the addition of ascorbate oxidase, allowing for differential measurements on an undiluted, untreated wine sample that corresponded well with commercial l ‐malate testing kits. Overall, this work demonstrates the power of an enzyme‐centric approach for designing electrochemical biosensors with improved operational parameters and novel functionality.