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Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20.
- Source :
-
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2016 Apr; Vol. 100 (8), pp. 3567-75. Date of Electronic Publication: 2015 Dec 11. - Publication Year :
- 2016
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Abstract
- Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of "blockbuster" drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.
- Subjects :
- Acetoacetates chemistry
Biocatalysis
Chryseobacterium chemistry
Chryseobacterium genetics
Enzyme Stability
Hot Temperature
Isomerism
Kinetics
Mutation
Oxidoreductases chemistry
Acetoacetates metabolism
Bacterial Proteins genetics
Bacterial Proteins metabolism
Chryseobacterium enzymology
Oxidoreductases genetics
Oxidoreductases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1432-0614
- Volume :
- 100
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Applied microbiology and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 26658823
- Full Text :
- https://doi.org/10.1007/s00253-015-7200-2