Figure 9. Transferability of the improved expression system to further eukaryotic PPOs.

Further eukaryotic PPOs (s. Table 1) were cloned and heterologously expressed in E. coli Rosetta 2 (DE3) [pLysSRARE2]. Production cultures were grown with different concentrations (0 mM, 0.02 mM, 0.2 mM) of supplemented CuCl₂ in AIM. A SDS-PAGE of purified proteins. Cultures were harvested after 48 h and recombinant PPOs were purified via affinity-chromatography using Strep-Tactin. Equal protein amounts were loaded on an acrylamide gel and analyzed via SDS-PAGE using Coomassie staining. B Specific PPO-activity after purification. Activity (v₀) was analyzed per mg purified protein for 4-methylcatechol as substrate. Purified ToPPO-2 produced with 0.2 mM CuCl₂ supplementation served as positive control (+) and buffer as negative control (-).