2.3. Monitoring of Pyroglutamate Conversion in Crude Cell Extract

Just before the cultures reached maximal optical density, a culture volume corresponding to 10 mg cell dry weight was harvested by centrifugation (12,000 ×g, 5 min, 4°C) and resuspended in 500 μL 10 mM HEPES buffer (pH 6.5). Cells were disrupted by sonication thrice with 1 min pulse and 2 min of cooling. Cell debris was removed by centrifugation, and the supernatant was used as crude cell extract. 100 μL of crude cell extract was heated at 65°C for 24 h in 10 mM HEPES buffer containing 2.2 mM L-pyroglutamate and 1 mM MgCl₂ supplemented with or without 14 mM ATP (pH 6.5) (total volume of 1 mL). As control, 2.2 mM L-pyroglutamate and 14 mM ATP were heated at 65°C for 24 h in 10 mM HEPES buffer supplemented with 1 mM MgCl₂ (pH 6.5). After heating, proteins were precipitated by chloroform and removed by centrifugation (20,000 ×g, 5 min, 4°C), and the supernatant was analysed for glutamate and pyroglutamate content. Protein concentration of the crude cell extract was determined using the Bicinchoninic acid Protein Assay Kit (Sigma-Aldrich, Germany) following the manufacturer's instructions. Enzymatic activity was calculated based on the difference between the initial pyroglutamate concentration (added pyroglutamate and pyroglutamate of the cell crude extract (CE control)) and the pyroglutamate concentration after 24 h of incubation (CE –/+ ATP) and correlated to a control without cell crude extract (Glp control) and to the protein content of cell crude extract. Quantification of ATP was done using the BacTiter-Glo™ Microbial Cell Viability Assay from Promega (Madison, WI, USA).