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

Ivana Drienovská, Matúš Gajdoš, Alexia Kindler, Mahsa Takhtehchian, Barbara Darnhofer, Ruth Birner-Gruenberger, Mark Dörr, Uwe T. Bornscheuer, Robert Kourist*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)12338-12342
Number of pages5
JournalChemistry - A European Journal
Volume26
Issue number54
DOIs
Publication statusPublished - 25 Sep 2020

Keywords

  • biocatalysis
  • enzyme expression
  • noncanonical amino acids
  • protein engineering
  • pseudomonas fluorescens esterase

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