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Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.
- Source :
-
Biotechnology and bioengineering [Biotechnol Bioeng] 2024 May; Vol. 121 (5), pp. 1532-1542. Date of Electronic Publication: 2024 Jan 24. - Publication Year :
- 2024
-
Abstract
- Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCR <subscript>M4</subscript> (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCR <subscript>M4</subscript> -V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCR <subscript>M4</subscript> . Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.<br /> (© 2024 Wiley Periodicals LLC.)
Details
- Language :
- English
- ISSN :
- 1097-0290
- Volume :
- 121
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Biotechnology and bioengineering
- Publication Type :
- Academic Journal
- Accession number :
- 38265115
- Full Text :
- https://doi.org/10.1002/bit.28662