| Autor: |
Smutok OV; Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine. smutok@cellbiol.lviv.ua.; Drohobych Ivan Franko State Pedagogical University, Drohobych, Ukraine. smutok@cellbiol.lviv.ua.; Clarkson University, Potsdam, NY, USA. smutok@cellbiol.lviv.ua., Dmytruk KV; Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine., Kavetskyy TS; Drohobych Ivan Franko State Pedagogical University, Drohobych, Ukraine.; The John Paul II Catholic University of Lublin, Lublin, Poland., Sibirny AA; Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine.; University of Rzeszow, Rzeszow, Poland., Gonchar MV; Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine. |
| Abstrakt: |
Flavocytochrome b 2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b 2 ) 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 2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b 2 , 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 2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b 2 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. |
