Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20

Abstract

Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)is an important chiral intermediate for the synthesis of Bblockbuster^drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bioreductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of Ch-KRED20 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.

Efficient Asymmetric Synthesis of Ethyl (S)-4-Chloro-3-hydroxybutyrate Using Alcohol Dehydrogenase SmADH31 with High Tolerance of Substrate and Product in a Monophasic Aqueous System

“…For the substrate COBE, we observed that the K m value of SmADH31 was only 0.42 mM, which was lower than that of other ADHs including ChADH (7.5 mM) and ScADH (0.59 mM). 21,22 The SmADH31 k cat /K m value for COBE was 5.5 × 10 2 mM −1 s −1 , which was higher than that of ChADH (26.8 mM −1 s −1 ) and ScADH (34.7 mM −1 s −1 ). All these results indicate that SmADH31 possesses a strong binding affinity and high catalytic efficiency for COBE.…”

Preparation of chiral aryl alcohols: a controllable enzymatic strategy via light-driven NAD(P)H regeneration

“…We selected the ketoreductase Ch KRED20 (cofactor NADH), with satisfactory ( R ) selectivity, 38 for screening reaction as a model enzyme. Given the optimized reaction conditions, we investigated the range and versatility of this method.…”

Stereoselective bioreduction of ethyl 3-oxo-3-(2-thienyl) propanoateusing theshort-chain dehydrogenase/reductaseChKRED12

“…Enantiomerically pure alcohols are known as the important and valuable chiral synthons for the production of pharmaceutical and fine chemicals [39][40][41]. To investigate the substrate specificity of ChKRED12, some substrates (Table 2, 1a-7a, 10 mM) were tested.…”