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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.