Department of Analytical Biotechnology, Institute of Cell Biology, NAS of Ukraine, Lviv, Ukraine.
Department of Biology and Chemistry, and Department of Physics of Drohobych Ivan Franko State Pedagogical University, Drohobych, Ukraine.
Yeast. 2019 May;36(5):341-348. doi: 10.1002/yea.3372. Epub 2018 Dec 21.
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 b , 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 ·m ), 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.
在最近几十年中，积累了大量关于 D-乳酸在食品技术和质量控制中的重要性的数据。如今，特别是在生物传感器方面，开发用于测定 D-乳酸的新方法非常相关。为了构建 D-乳酸选择性生物传感器，我们建议使用重组酵母细胞的线粒体，该酵母细胞来自 Ogataea（Hansenula）polymorpha“tr6”（gcr1 catX/Δcyb2，prAOX_DLDH），该酵母细胞过量产生 D-乳酸：细胞色素 c-氧化还原酶（DLDH，EC 1.1.2.4），并且缺乏 L-乳酸特异性酶（黄素细胞色素 b，EC 1.1.2.3）。由于 DLDH 的跨膜定位，纯酶的使用存在问题，因为其分离和稳定化很复杂。该酶催化 D-乳酸氧化为丙酮酸，同时将铁细胞色素 c 还原为亚铁细胞色素 c。构建的生物传感器具有高灵敏度（18.5 А·М ·m）、低检测限（3 μM 的 D-乳酸）、宽线性范围、良好的选择性和足够的稳定性。对乳制品中的 D-乳酸进行了实际样品分析，并且与参考方法（0.7 < r < 1）和文献数据具有高度相关性。
