Том 74, № 4 (2019)

The oxidation of cephalosporin C (CPC) by D-amino acid oxidase is the first stage in the biocatalytic industrial process of the production of 7-aminocephalosporanic acid, the initial synthon for the production of semisynthetic cephalosporin antibiotics. We determined the kinetic parameters (catalytic constant rate and Michaelis constant) of the wild-type recombinant oxidase from the yeast Trigonopsis variabilis (TvDAAO); three mutant forms of the enzyme with point Met-to-Leu substitutions at positions 104, 156, and 209; and the TvDAAO RDAF quadruple mutant using the dependence of the initial reaction rate on the CPC concentration (the initial rate method). The point substitutions in the mutants are shown to lead primarily to a decrease in the Michaelis constant. The largest (39%) increase in catalytic efficiency k_cat/K_M is observed for the mutant with the Met156Leu substitution. The Michaelis constant for the increased four times compared to that for the wild-type enzyme in the reaction with CPC, whereas the catalytic efficiency remains almost unchanged due to a four-fold increase in the catalytic rate constant.

Cell-based reporters expressing luciferase fusions with transcription factors or their protein stability domains can be considered as microbioreactors providing a way to directly monitor the stability of the luciferase labeled transcription factor or its domain in real-time. To understand principal advantages and/or limitations of these systems for the purposes of applied and fundamental research one needs to develop a quantitative description of their performance based on the determination of actual intracellular concentrations of the fusion proteins and rates of their production. In this work, the experimental data generated by means of luciferase activity calibration were used to calculate the steady-state intracellular concentrations of luciferase fusions in SH-SY5Y neuroblastoma cell lines stably expressing HIF1 ODD-luc and Neh2-luc proteins. For both reporters, the concentration of fusion proteins was determined as 60–80 nM, the values close to those for Michaelis constants for HIF prolyl hydroxylase (10–100 nM HIF) and the dissociation constant for Keap1-Nrf2 complex (50 nM), the parameters controlling rate-limiting steps of HIF1 ODD-luc and Neh2-luc reporter performance, respectively. New data allowed us to calculate the production rates and maximum concentrations for the fusion proteins under the conditions of irreversible activation and protein stabilization. The quantitative analysis of the Neh2-luc reporter performance employing the newly generated parameters explains the multi-order shift in the apparent activation constant versus the “real” dissociation constant determined for a known Nrf2 displacement activator using fluorescent polarization homogeneous assay with recombinant Keap1 and labeled Nrf2 peptide.

The Bacille Calmette–Guerine (BCG) vaccine, which is based on a live strain of Mycobacterium bovis BCG is widely used for the immunoprophylaxis of tuberculosis. One of the BCG vaccine’s key parameters is its сell viability (specific activity: the number of colony forming units, CFUs), which is traditionally defined by the microbiological method. In this work, the rapid and selective bioluminescent method of intracellular ATP assay is used to control the BCG vaccine’s viability at various stages of the vaccine’s production. It permits us to reduce the time required for the analysis from 28 days to 1 h. The correlation is shown between the viability of a liquid BCG vaccine measured by the microbiological method compared to one calculated using the content of intracellular ATP, as well as the correlation between the CFU value for the lyophilized BCG vaccine and the ATP content in the liquid vaccine before lyophilization.

An open, single, crossover, pharmacokinetic study of GML-1 (dose 50 mg/kg) in rabbits after its oral administration in tablets and alone as a substance is carried out. The following pharmacokinetic parameters are calculated: the maximum concentration of GML-1 in blood plasma (C_max) of rabbits, the time required to reach C_max, the area under the concentration-time curve, the half-time of GML-1, and the relative bioavailability. The GML-1 concentration is detected with the use of the HPLC-MS (ion trap) by the daughter ions. The relative bioavailability of GML-1 in tablets is 101.72 ± 19.96% in comparison to the substance.