z-logo
Premium
Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization
Author(s) -
Minges Hannah,
Schnepel Christian,
Böttcher Dominique,
Weiß Martin S.,
Sproß Jens,
Bornscheuer Uwe T.,
Sewald Norbert
Publication year - 2020
Publication title -
chemcatchem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.497
H-Index - 106
eISSN - 1867-3899
pISSN - 1867-3880
DOI - 10.1002/cctc.201901827
Subject(s) - thermostability , directed evolution , protein engineering , mutagenesis , chemistry , rational design , enzyme , mutant , active site , combinatorial chemistry , biochemical engineering , computational biology , biochemistry , biology , genetics , engineering , gene
Halogenases are valuable biocatalysts for selective C−H activation, but despite recent efforts to broaden their application scope by means of protein engineering, improvement of thermostability and catalytic efficiency is still desired. A directed evolution campaign aimed at generating a thermostable flavin‐dependent tryptophan 6‐halogenase with reasonable activity suitable for chemoenzymatic purposes. These characteristics were tackled by combining successive rounds of epPCR along with semi‐rational mutagenesis leading to a triple mutant (Thal‐GLV) with substantially increased thermostability (▵T M =23.5 K) and higher activity at 25 °C than the wild type enzyme. Moreover, an active‐site mutation has a striking impact on thermostability but also on enantioselectivity. Our data contribute to a detailed understanding of biohalogenation and provide a profound basis for future engineering strategies to facilitate chemoenzymatic application of these attractive biocatalysts.

This content is not available in your region!

Continue researching here.

Having issues? You can contact us here