A Study On The Inactivation Mechanism And Stabilization Of Aldo-keto Reductase YtbE

Bioreduction of methyl o-chlorobenzoylformate by aldo-keto reductase YtbE showed great potential to be a green and cost-effective way to produce methyl (R)-o-chloromandelate, a key intermediate for a platelet aggregation inhibitor, (S)-clopidogrel. However, the practical use of YtbE, a very competitive and promising candidate, has been largely hampered by its rather poor thermostability and operational stability.Here we report a comprehensive strategy to improve the bioreduction by stabilizing YtbE based on the inactivation mechanism study. Biochemical analysis demonstrated that the inactivation of YtbE involves a dominating unfolding step followed by an irreversible dimerization step induced by intermolecular disulfide bonds. The proposed inactivation model showed a high applicability to describe the inactivation process of YtbE. Based on the inactivation mechanism, approaches to inhibiting the two inactivation steps were proposed and confirmed. Glycerol and other additives were used to suppress the protein unfolding while dithiothreitol (DTT) was introduced to inhibit dimerization. The engineered YtbE showed great improvement in thermal and operational stability so the conversion of 100 mM CBFM reduction by YtbE powders and GDH powders was greatly increased from 62.9% to 98.7%. Moreover, the conversion of 1 M CBFM reduction by lyophilized cells of recombinant E. coli (YtbE/GDH) was also increased from 70.5% to 96.6% without additional cofactors. Furthermore, the half-life of YtbE in the practical reaction was significantly increased from 46.6 min to 158.7 min. Therefore, this work takes an important step towards the commercialization of YtbE in the pharmaceutical industry.