Thermomixer comfort 5355

An amount of 2 mL bioreactor liquid was taken, centrifuged (3200× g, 10 min, 4 °C), and the pellet was dissolved in 1 mL lysis buffer (10 mM Tris-HCl, NaCl 2 mg/mL, 1 mM PMSF, 4 mg/mL SDS). Bacteria were disrupted by bead beating (FastPrep-24, MP Biomedicals, Sanra Ana, CA, USA, 5.5 ms, 1 min, 3 cycles) followed by 15 min at 60 °C (Thermomixer comfort 5355, Eppendorf, Hamburg, Germany) and ultra-sonication (UP50H, Hielscher, Teltow, Germany, cycle 0.5, amplitude 60%). Protein concentration was determined with the bicinchoninic acid assay, according to the user manual (Pierce™ BCA Protein Assay Kit, Thermo Fischer Scientific, Waltham, MA, USA). Furthermore, 100 µg of protein was precipitated overnight at −20 °C with ice-cold acetone 1:5 (v/v) and centrifuged for 10 min at 14,000× g. The pellet was used for sodium dodecyl sulfate poly acryl amid one-dimensional gel electrophorese (SDS–PAGE). SDS-PAGE analysis, in-gel digestion, and protein purification with ZipTip® treatment were performed [36 (link)].

OMT activity of TaBX10 was tested using enzyme assays containing 0.5 mM dithiothreitol, 0.5 mM S-adenosyl-l-methionine, substrate (DIMBOA-Glc + DIM₂BOA-Glc; 25 μg ml⁻¹), and 35 μl of desalted enzyme in a volume of 100 μl. The assays were incubated overnight in glass vials at 25°C at 300 rpm using a ThermoMixer comfort 5355 (Eppendorf). The reaction was stopped with 1 volume 100% methanol and centrifuged for 5 min at 5000g. Product formation was monitored by the analytical methods described above.