## 3 Results
### Cultivation of Recombinant Protein-Producing Cells in Media Containing PFCs
These results differ from those that are published in other works [13; 15], where the optimum concentration of PFD in the medium for cultivating _E. coli_ cells was 5% or 50% (\(v/v\)). Thus, using PFH and polyethers for obtaining recombinant proteins from _E. coli_ cells was more efficient as compared to PFD, because the maximum of the target active enzyme quantity was achieved at relatively low concentration of PFCs additive to the culture medium. \begin{table}
\begin{tabular}{c c c c c c} \hline
**PFC (\% v/v)** & **0.3** & **0.5** & **1** & **3** & **5** \\ \hline PFH & 0.363 \(\pm\) 0.015 & 0.368 \(\pm\) 0.017 & 0.374 \(\pm\) 0.017 & 0.351 \(\pm\) 0.014 & 0.349 \(\pm\) 0.013 \\ PFD & 0.345 \(\pm\) 0.012 & 0.345 \(\pm\) 0.012 & 0.345 \(\pm\) 0.012 & 0.346 \(\pm\) 0.012 & 0.345 \(\pm\) 0.012 \\ Polyether I & 0.352 \(\pm\) 0.014 & 0.357 \(\pm\) 0.015 & 0.358 \(\pm\) 0.014 & 0.353 \(\pm\) 0.014 & 0.347 \(\pm\) 0.012 \\ Polyether II & 0.367 \(\pm\) 0.016 & 0.369 \(\pm\) 0.016 & 0.376 \(\pm\) 0.017 & 0.352 \(\pm\) 0.014 & 0.349 \(\pm\) 0.013 \\ \hline \end{tabular} \({}^{\star}\) Specific cell growth rate in the absence of PFCs was 0.344 \(\pm\) 0.011 h\({}^{-1}\).  
\end{table} 
Table 2: Specific cell growth rate (\(\mu\), h\({}^{-1}\)) for _E. coli_ SG13009[pREP4] for various concentrations of PFCs in the culture medium \({}^{\star}\). Figure 1: The accumulated biomass of _E. coli_ SG13009[pREP4] cells (**a**) and His\({}_{6}\)-OPH-activity in the cells (**b**) vs. initial concentration of the additive (\(\blacksquare\)–FFH, \(\blacktriangle\)–polyether I, \(\blacktriangle\)–PFD, \(\blacklozenge\)–polyether II) Control sample (without PFC additives) is marked by dashed line. It was established that the highest total His\({}_{6}\)-OPH activity per 1 L of culture medium was provided by the two substances, PFH and polyether II, which have the highest O\({}_{2}\) solubility at 298 K among the PFCs studied (Table 1). The evaluated total His\({}_{6}\)-OPH activity of the accumulated cells per 1 L of culture medium was higher by 11% in the presence of polyether II than that in the presence of PFH. However, the latter PFC, being a commercially available product with strictly standardized characteristics, appears to be the better choice for practical applications involving the biosynthesis of recombinant proteins. Taking this into consideration, we used PFH with a concentration of 0.5 vol. % in our further experiments. Studying the Possibility of muL triple re-Use of Gas Transport-Capable Substances in Biosynthesis of Intracellular Proteins  
When PFCs are used for cultivation of microorganisms with exoproducts, the fluoroorganic phase becomes separated from the culture medium upon the termination of the process and precipitation of the compound to the bottom of the reactor [12]. Since His\({}_{6}\)-OPH is an intracellular enzyme, re-use of gas transport-capable substances in the biosynthesis of this enzyme requires an essentially different approach. In order to obtain the target enzyme, the His\({}_{6}\)-OPH-containing biomass of _E. coli_ SG13009[pREP4] cells that accumulated during cultivation in the presence of 0.5 vol. % of PFH was disintegrated in the presence of phosphate buffer, and the resulting supernatant was used for protein isolation, in accordance to standard techniques [32]. The remaining precipitate of cell debris, being in fact a mixture of disintegrated cell biomass and PFH, was added to the TYE culture medium instead of the pure fluorocarbon and 1/4 of yeast extract, which were used for the 1-st biotechnological round of biomass accumulation (Figure 2). Figure 2: General scheme of the recycling process of perfluorohexane (PFH) present in the disintegrated biomass.