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

Abstract

Protein design is limited by the diversity offunctional groups providedbythe canonical protein"building blocks". Incorporating noncanonical amino acids(ncAAs)into enzymes enablesadramatic expansionoftheir catalytic features.For this, quick identification of fullytranslated and correctly folded variantsisdecisive. Herein,we report the engineering of the enantioselectivity of anesteraseutilizing several ncAAs. Key for the identificationof active and soluble protein variants was the use of thesplit-GFP method,which is crucial as it allowssimple de-termination of the expression levels of enzyme variantswith ncAA incorporationsbyfluorescence. Several identi-fied variants led to improved enantios electiv ityoreven in-verted enantiopreference in the kinetic resolution of ethyl3-phenylbutyrate.