Fig. 3: Directed evolution of P450BM3 for N-oxidation of N-heterocycles.

a Mutant library screening. The reactions were conducted in a 96-well plate, with each well containing 200 μL a K₂HPO₄/KH₂PO₄ buffer (50 mM, pH 8.0) with quinoline (1 mM), crude enzyme solution (100 μL), 1 mM NADPH, and was incubated at 30 °C for 10 min. The absorbance at 340 nm (OD₃₄₀) was then measured. The red line indicates the threshold used for the selection of mutants for further characterization. 96 variants are tested in per site, n = 96 independent experiments. Source data are provided as a Source Data file. b Conversion and N-oxidation selectivity of the positive mutants in site-directed saturation mutations determined by HPLC. The reactions were carried out in 1.5 mL Eppendorf tubes, each containing 1 mL of a K₂HPO₄/KH₂PO₄ buffer with quinoline (1 mM), crude enzyme (960 µL), and 2 mM NADPH. The reactions were incubated at 30 °C for 16 h. Reactions were conducted in three independent biological experiments (n = 3). The data are presented as mean values ± SD across three independent repetitions. Error bars indicate the standard deviation of the replicate measurements, with the error bar centers representing the means of these replicates. Replicate measurements are depicted as block circles. The source data are provided in a Source Data file.