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. 2021:2280:249-260.
doi: 10.1007/978-1-0716-1286-6_16.

Flavocytochrome b2 of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application

Oleh V Smutok  1   2   3 , Kostyantyn V Dmytruk  4 , Taras S Kavetskyy  5   6 , Andriy A Sibirny  4   7 , Mykhailo V Gonchar  4
Affiliations

Flavocytochrome b2 of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application

Oleh V Smutok et al. Methods Mol Biol. 2021.

Abstract

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) 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 b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, 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 b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 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.

Keywords: Amperometric biosensor; CYB2; Enzymatic kits; Enzyme purification; Flavocytochrome b2; L-lactate analysis; Ogataea polymorpha; Overproducers.

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