l-lactate sensors by a single amino acid mutation in Aerococcus viridans l-lactate oxidase]]>

摘要

Abstractl-lactate biosensors employingl-lactate oxidase (LOx) have been developed mainly to measurel-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Somel-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection ofl-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 inAerococcus viridansLOx (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 0.5mMl-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 c