Premium
Engineering the Active Site of an ( S )‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines
Author(s) -
Land Henrik,
Ruggieri Federica,
Szekrenyi Anna,
Fessner WolfDieter,
Berglund Per
Publication year - 2020
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201901252
Subject(s) - chemistry , amine gas treating , kinetic resolution , active site , substrate (aquarium) , transaminase , primary (astronomy) , combinatorial chemistry , catalysis , optically active , protein engineering , reagent , enzyme , stereochemistry , organic chemistry , enantioselective synthesis , oceanography , physics , astronomy , geology
A protein engineering approach for expanding the substrate scope of the ( S )‐selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl‐ and benzyl‐substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2‐diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure ( R )‐1,2‐diphenylethylamine with ee >99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20‐30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published ( S )‐selective amine transaminase showing activity towards benzyl‐substituted primary amines.