Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.
Drohobych Ivan Franko State Pedagogical University, Drohobych, Ukraine.
Methods Mol Biol. 2021;2280:249-260. doi: 10.1007/978-1-0716-1286-6_16.
Flavocytochrome b (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.
黄素细胞色素 b（EC 1.1.2.3；L-乳酸：细胞色素 c 氧化还原酶，FC b）来源于耐热甲基营养酵母 Ogataea polymorpha，是一种耐热酶，有望在医学、营养领域以及商业产品的质量控制中进行高度选择性的 L-乳酸分析。在这里，我们描述了通过过表达编码 FC b 的 CYB2 基因 O. polymorpha，在质粒框架下在强酒精氧化酶启动子的控制下构建 FC b 产生菌，用于多拷贝整合，随后转化受体制剂 O. polymorpha C-105（gcr1 catX），该受体制剂在葡萄糖抑制作用下受损且缺乏过氧化氢酶活性。选择的重组菌株 O. polymorpha“tr1”（gcr1 catX CYB2）与初始菌株相比，FC b 活性增加了 8 倍，被用作酶的来源。为了纯化 FC b，开发了一种新的亲和层析方法，并使用纯化的酶制剂用于构建高度选择性的酶试剂盒和用于分析人体液体和食品中 L-乳酸的电流型生物传感器。
