Cooperative Enhancement of Aldoxime Dehydratase Stability through Whole‐Cell Immobilization and Flow Reactor Integration

This study investigates the synthesis of aromatic nitriles using an evolved variant of OxdF1 (L318F/F306Y), an aldoxime dehydratase from Pseudomonas putida F1, engineered for improved catalytic efficiency toward benzaldehyde oxime. The double OxdF1 (L318F/F306Y) mutant effectively catalyzes the conversion of various benzaldoxime derivatives to the corresponding nitriles. Due to the enzyme's inherent instability, immobilized whole-cell systems are employed in a flow reactor to improve its stability and broaden its applicability, with the biotransformation of benzaldehyde oxime and 2,6-difluorobenzaldehyde oxime serving as case studies. The enzyme's stability is markedly improved, maintaining 87% yield even after 8 h of processing in the preparation of benzonitrile. Preparation of 2,6-difluorobenzontirile poses additional challenges due to the low water solubility of both the substrate, and even more so, the product, an important intermediate in various chemical applications. To overcome solubility limitations, a segmented liquid–liquid flow system (water/cyclohexane) was implemented, significantly improving the enzyme stability. The process was run continuously for 12 h, with a conversion of ≈70% by the end of the operation. Furthermore, 2,6-difluorobenzonitrile is selectively extracted in-line using a liquid–liquid extractor, thus, facilitating its efficient recovery and purification.