D-lactate-selective amperometric biosensor based on the mitochondrial fraction of Ogataea polymorpha recombinant cells.

During the recent decades, a lot of data about the significance of D-lactate determination in food technology and quality control have been accumulated. Nowadays, the development of new methods for the determination of D-lactate is very relevant, especially with regard to biosensors. To construct a D-lactate-selective biosensor, we suggest using the mitochondria of recombinant yeast cells of Ogataea (Hansenula) polymorpha "tr6" (gcr1 catX/Δcyb2, prAOX_DLDH) overproducing D-lactate: cytochrome c-oxidoreductase (DLDH, EC 1.1.2.4) and lacking an L-lactate-specific enzyme (flavocytochrome b2 , E.C. 1.1.2.3). The usage of the pure enzyme is problematic due to the complexity of its isolation and stabilization because of the intramembranous localization of DLDH. The enzyme catalyzes D-lactate oxidation to pyruvate coupled with ferricytochrome c reduction to ferrocytochrome c. The constructed biosensor is characterized by high sensitivity (18.5 А·М-1 ·m-2 ), a low detection limit (3 μM of D-lactate), wide linear ranges, good selectivity, and sufficient stability. The real samples' analysis of D-lactate in dairy products was performed, and high correlation of the obtained results with the reference approach (0.7 < r < 1) and literature data was demonstrated.