[Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)].

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.

[Screening of yeasts producing stable L-lactate cytochrome c oxidoreductase and regulation of enzyme synthesis].

Screening of strains producing a stable form of L-lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) was carried out among 14 yeast species. Enzyme activity was detected in polyacrylamide gel after the electrophoresis of cell-free extracts. The FC b2 of Hansenula polymorpha, Rhodotorula pilimanae, and Kluyveromyces lactis are characterized by high thermostability; in particular, the FC b2 of H. polymorpha retains its activity and tetrameric structure even after heating at 60 degrees C for 10 min. Constitutive synthesis of FC b2 was observed in H. polymorpha grown on either glucose, ethanol, or glycerol. L-Lactate induces de novo synthesis of FC b2, as proved by the use of cycloheximide, an inhibitor of protein synthesis.

[New method for detection of enzymatic activity of flavocytochrome b2 in electrophoregrams]. / Novyi metod obnaruzheniia fermentativnoi aktivnosti flavotsitokhroma b2 na élektroforegrammakh.

A new method of visualization of the activity of flavocytochrome b2 (FC b2; L-lactate: ferricytochrome c oxidoreductase, EC 1.1.2.3) in electrophoretograms was developed, based on the interaction between ferrocyanide (generated during the enzymatic reaction) and Fe23+, resulting in the formation of intensely colored precipitates of Berlin blue. The main advantages of this method were its high sensitivity (less than 0.005 U FC b2 was detected within a suitable time period) and the stability of the dye formed. The method developed can be used for determining FC b2 activity in cell-free extracts (e.g., in the selection of FC b2 producers) and monitoring chromatographic purification of proteins, as well as in other cases associated with FC b2 assessment.