Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional‐Group Diversity for Enzyme Engineering

Protein design is limited by the diversity of functional groups provided by the canonical protein „building blocks“. Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split‐GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3‐phenylbutyrate. In enzyme engineering using an expanded genetical code, quick identification of fully translated and correctly folded variants is crucial. Herein, we report the engineering of the aryl esterase from Pseudomonas fluorescens utilizing a pool of ncAAs. Key for the identification of active and soluble protein variants was the use of the split‐GFP method, allowing for quick identification of interesting variants.