초록
Asymmetric reduction of methyl o-chlorobenzoylformate (CBFM) using aldo-keto reductase YtbE is a potentially cost-effective and green technology in manufacturing methyl ®-o-chloromandelate which is a key intermediate for synthesizing (S)-clopidogrel (a popular medicine for treating atherosclerosis). At the moment, large scale application of YtbE has been complicated by uncertain thermal and operational stabilities. Consequently, we endeavored possible enzyme inactivation mechanism, and showed that (a) unfolding of YtbE explains enzyme activity loss, and (b) YtbE dimerization has a less significant effect owing to a small quantity detected. The effects of substrate and temperature on YtbE are mostly upheld by a one-step inactivation model, whereas the effect of product by a 2-step activity reduction modality. Partially based on these new understandings, a multi-factor experimental strategy was rationalized for improving the YtbE stability. For instance, glycerol was introduced to reduce enzyme unfolding whilst dithiothreitol to suppress its dimerization. This improved substrate conversion from 62.9% to 98.7%, and from 70.5% to 96.6% at 0.1M and 1.0M CBFM, respectively, with YtbE half-life being increased from 46.6min to 159min.