Berberine bridge enzyme (BBE) catalyses the oxidative formation of an intramolecular CC bond using (S)-reticuline as the natural substrate to form (S)-scoulerine as the product. To allow application of the enzyme on a preparative scale for the synthesis of novel optically pure berbine and isoquinoline derivatives, an organic solvent is required to solubilise the barely soluble substrates. It was shown that BBE tolerates a broad variety of organic co-solvents. Ideally the enzymatic enantioselective oxidative CC bond formation can be performed in 70% v v−1 toluene concentration, which allowed a soluble substrate concentration of at least 20 g L−1. In addition, the enzyme works in a broad operational window concerning pH and temperature. High conversions can be reached between pH 8 and 11 and from 30 to 50 °C, respectively. The enantioselective oxidative CC bond formation was demonstrated on a preparative scale (500 mg) in a kinetic resolution leading to optically pure products (>97% ee).