Structural-guided design to improve the catalytic performance of aldo-keto reductase KdAKR.
Biotechnol Bioeng
; 120(12): 3543-3556, 2023 Dec.
Article
en En
| MEDLINE
| ID: mdl-37641876
ABSTRACT
Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t1/2 ) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure-function relationship of AKRs, providing a certain guidance for the modification of other AKRs.
Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Kluyveromyces
/
Caproatos
Idioma:
En
Revista:
Biotechnol Bioeng
Año:
2023
Tipo del documento:
Article