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Screening for stereoselective cyanohydrin synthesis in 96-well plates was employed in the development of an efficient, pH-stable hydroxynitrile lyase for the conversion of sterically hindered aliphatic aldehydes. Site-saturation mutagenesis (SSM) resulted in a powerful catalyst for the stereoselective conversion of hydroxypivalaldehyde and pivalaldehyde to their corresponding (R)-cyanohydrins (ee > 97%) which are used as chiral building blocks (e.g., for pantothenic acid production). Furthermore, redesigning the PaHNL5 gene and improving its expression by Pichia pastoris with the help of a new P AOX1 promoter variant and the helper protein PDI (protein disulfide isomerase) led to elevated amounts of todays most efficient biocatalyst for vitamin B 5 synthesis.

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Structure‐guided redesign of the active site of almond (R)‐PaHNL5 for increased enantioselectivity resulted in four improved muteins. In particular, mutation V360I gave enhanced conversion rates and enantioselectivities higher than 96 % ee for two structurally related substrates 1 and 2. Chiral building blocks for the synthesis of pharmaceutically active “prils” (example shown) can thus be produced on a large scale.