Directed Evolution Of Mandelate Racemase Based On A Novel High-throughput Screening Method

Optically pure methyl(R)-o-chloromandelate and(R)-acetyl-o-mandelic acid are key intermediates for the industrial synthesis of(S)-clopidogrel,which could be prepared with 100%theoretical yield by dynamic kinetic resolutions(DKR)using a proper esterase and racemase.Enzymatic DKR does not need harsh chemical conditions and generates little waste,which accords with the target of green chemistry.However,efficient enzymatic DKR is hindered by the low catalytic activity of mandelate racemase(MR)toward(S)-o-chloromandelic acid.In the present work,we proposed to improve the catalytic performance of MR toward(S)-2-CMA by directed evolution and developed a novel method for high-throughput screening(HTS)of MR libraries.To facilitate directed evolution of MR,we developed an enantioselective oxidation system for HTS of mutant libraries.In this assay,an(R)-enantioselective mandelate dehydrogenase from Rhodotorula graminis was used to convert the(R)-enantiomer produced from racemization reaction to keto acid for further colorimetric reactions.Subsequently,the accuracy of the developed HTS method was validated by chiral HPLC analysis.The results indicated that the developed colorimetric HTS method was applicable for directed evolution of MR.Based on this HTS method,a triple mutant V22I/V29I/Y54F(MRDE1)with 3-fold greater relative activity as compared to the native MR was obtained through a combined strategy of directed evolution and rational design.Meanwhile,kinetic analysis was conducted for the wild-type and mutants of MR with both enantiomers to explore the cause for the increased activity.The kinetic data not only revealed the occurrence of synergistic effect among mutation sites,but also suggested that the enhanced catalytic efficiency mainly arose from the elevated kcat.Finally,further insight into the source of improved catalytic activity was revealed by molecular docking and molecular dynamic simulations.It was found that the enhancement of protolysis and the introduction of?-?interaction between thesubstrate and substituted residues contributed to the improved catalytic performance.Furthermore,molecular mechanics/Poisson-Boltzmann surface area analysis was employed to investigate the binding free energy of enzyme-substrate complexes.The results suggested that the increase in the electrostatic contribution might be the major reason for the decrease in binding free energy of MRDE1,which indicated that the electrostatic force may play an important role in the interaction network of the active center.The results demonstrate that directed evolution is a useful and powerful means to generate enzymes with improved enzymatic properties,with the development of an appropriate HTS method as the premise.Moreover,the HTS method developed in this work and the successful directed evolution of MR based on this method provide an example for racemase engineering,and may inspire directed evolution of other racemases toward enhanced catalytic performance on non-natural substrates.