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...

Full description

Saved in:
Bibliographic Details
Published in:Chemistry : a European journal 2020-09, Vol.26 (54), p.12338-12342
Main Authors: Drienovská, Ivana, Gajdoš, Matúš, Kindler, Alexia, Takhtehchian, Mahsa, Darnhofer, Barbara, Birner‐Gruenberger, Ruth, Dörr, Mark, Bornscheuer, Uwe T., Kourist, Robert
Format: Article
Language:English
Subjects:
Amino Acids
biocatalysis
Catalysis
Chemistry
Communication
Communications
Enantiomers
enzyme expression
Enzymes
Esterase
Esterases - chemistry
Esterases - metabolism
Fluorescence
Functional groups
Identification keys
noncanonical amino acids
Phenylbutyric acid
Protein Engineering
Proteins
pseudomonas fluorescens esterase
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0947-6539
1521-3765
DOI:10.1002/chem.202002077