Flavocytochrome b of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application.

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.