Sustainable Biocatalytic Synthesis of Chiral Cyanohydrins Using Engineered Hydroxynitrile Lyases

Enantiopure cyanohydrins are versatile chiral intermediates of several bioactive molecules and life-saving drugs. Consequently, the demand for their efficient and sustainable preparation has grown significantly. We aimed to synthesise them using native and engineered Arabidopsis thaliana hydroxynitrile lyase (AtHNL). Screening of the in-house AtHNL variant library created by saturation mutagenesis at F179 and Y14 using Feigl-Anger paper-based assay towards the synthesis of four distinct industrially important chiral cyanohydrins has uncovered nine variants with more promising activity than the wild-type. After optimisation of eight crucial biocatalytic parameters using acetone cyanohydrin with the model reaction of asymmetric hydrocyanation of benzaldehyde, the substrate scope of the variants was studied by converting 21 different aromatic aldehydes, among which 18 are newly investigated by these enzymes, into their corresponding valuable enantiopure (R)-cyanohydrins with good yields (up to 98%) and excellent optical purities (up to >99.9%). Kinetic studies of AtHNL variants revealed significant improvements, with a >27-fold increase in catalytic efficiency over the wild-type in the hydrocyanation of 4-allyloxybenzaldehyde. The improved catalytic performance was supported by molecular docking and simulation studies. These findings highlight the potential of AtHNL variants in enhancing activity, synthetic scope, and catalytic efficiency towards sustainable synthesis of industrially relevant chiral cyanohydrins.