Process intensification in chiral cyanohydrin synthesis: From biocatalyst engineering to bioreactor design

Enzymes are key green catalysts in the selective synthesis of high-value compounds. Their activity and stability depend not only on the enzyme's structure, but can also be modified by immobilisation method and type of reactors applied. In this context, carriers are often essential for the effective use of enzymes, and the chemical nature of the carrier surface can significantly impact the enzymes and reaction components. GtHNL-A40H/V42T/Q110H (GtHNL-3V) from Granulicella tundricola was therefore immobilised on silica carriers functionalised with amino, epoxy and n-octyl groups, including bifunctional carriers. Carriers with amino or epoxy groups enable the covalent binding of enzymes. Silica carriers with n-octyl groups are characterised by hydrophobic interactions, which, given the structure of GtHNL-3V, can stabilise the enzyme. The process was carried out in two types of reactors: batch and flow. It has been demonstrated that, depending on the type of reactor, bifunctionalised carriers can stabilise the enzyme or facilitate product release, resulting in higher reaction rates compared to monofunctional supports. It was also found that increasing protein loading improved enantioselectivity more effectively than lowering reaction temperature. In continuous flow, reaction volume was halved, and time was reduced from 1 h to 6 min, while minimising potential hydrogen cyanide release and reducing solvent consumption, thereby enhancing process safety and sustainability. These findings highlight the potential of bifunctional carriers for the efficient synthesis of chiral cyanohydrins, with broader applicability to other enzymatic transformations, including biphasic systems.