Second-Generation Engineering of a Thermostable Transketolase (TKGst ) for Aliphatic Aldehyde Acceptors with Either Improved or Reversed Stereoselectivity.
Chembiochem
; 18(5): 455-459, 2017 03 02.
Article
en En
| MEDLINE
| ID: mdl-28005308
ABSTRACT
The transketolase from Geobacillus stearothermophilus (TKGst ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TKGst based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TKGst for various applications in asymmetric carboligation.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Transcetolasa
/
Ingeniería de Proteínas
/
Aldehídos
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2017
Tipo del documento:
Article