TY  - JOUR
AB  - 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 degrees 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.
DA  - 2020
DO  - 10.1002/cctc.201901827
KW  - directed evolution
KW  - enzyme catalysis
KW  - enzyme stability
KW  - rational
KW  - mutagenesis
KW  - tryptophan halogenase
LA  - eng
IS  - 3
M2  - 818
PY  - 2020
SN  - 1867-3880
SP  - 818-831
T2  - ChemCatChem
TI  - Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29399331
Y2  - 2024-11-22T02:13:04
ER  -