Figure 2.

Development of a fed‐batch processing strategy for biocatalytic hydroxylation of C12:0. Top panel: Substrate feeding controlled by the steady‐state O₂ concentration in bulk liquid. The reaction (50 ml) contained 2 µM his_BM3 and 1.9 U/ml commercial GDH, 8 mM C12:0, 200 mM glucose, 500 µM NADP⁺, 1 mg/ml catalase, 3.5% dimethyl sulfoxide (DMSO; v/v), 50 mM potassium phosphate buffer (pH 7.5), and silicone antifoam. The reaction was operated at 25 ml/min O₂ gassing, 350 rpm magnetic stirring and automated pH control (pH 7.2). As indicated, new C12:0 (80 mg) dissolved in DMSO (1.25 ml) was added four times, resulting in a total concentration of 40 mM C12:0 and ~12% DMSO. Bottom panel: Fed‐batch reaction for hydroxylation of 80 mM C12:0. The reaction composition was identical to the reaction in the top panel, with the exception that 300 mM glucose and 2.8% DMSO (by volume) were used. Additionally, the feeding steps were adapted: five times 80 mg C12:0 in 0.92 ml DMSO and four times 100 mg C12:0 in 0.5 ml DMSO were added, resulting in a total concentration of 80 mM C12:0 and ~12% DMSO