Genome Mining And Characterization Of O-chloromandelate Racemase

(S)-2-chlorophenylglycine is an important non-natural chiral amino acid with biological activity,and also it is a building block of clopidogrel which can be used for the treatment of cardiovascnlar and cerebrovascular diseases.Racemic mandelic acid can be converted to optically pure(S)-phenylglycine currently through multi-enzyme cascade reactions consisting of mandelate racemase,leucine dehydrogenase and mandelate dehydrogenase.The cascade exhibited high efficiency,good enantioselectivity and satisficing atom economy.However,mandelate racemse showed low catalytic activity towards 2-chloromandelate,limiting its further application.This research focused on the identification of novel mandelate racemase and construction of racemase high-throughput screening method for the future directed evolution.With genome mining and molecular cloning,nine mandelate racemases(MRs)were identified in the study.A novel racemse named ArMR,which showed higher activity and better soluble protein expression,was isolated from Agrobacterium radiobacter.This enzyme was purified by affinity chromatography and the purified enzyme was used for futher characterization.ArMR displayed the optimum catalytic activity at 50℃,pH 7.8 in Tris-HCl buffer.The half-lives of ArMR at 50℃,40℃ and 30℃ are 0.17 h,27.2 h and 70.7 h.The KM values of ArMR towards(R)-mandelic acid and(S)-mandelic acid were 1.44 mM and 0.81 mM,respectively.The corresponding kcat values were 410 s-1 and 218 s-1.As a result,kcat/KM towards(R)-mandelic acid and(S)-mandelic acid were 284.6 mM-1s-1 and 269.5 mM-1s-1,respectively.In addition,the KM values of ArMR towards(R)-2-chloro mandelic acid and(S)-2-chloro mandelic acid were 6.48mM and 6.37 mM,and the corresponding kcat values were 0.22 s-1 and 0.23 s-1,respectively.Meanwhile,Mg2+and Mn2+were capable of activating the enzyme greatly.However,Zn2+ inactivated the enzyme completely.An enzyme-coupled colorimetric assay for mandelate racemae was constructed,which utilized a R-selective mandelate dehydrogenase named LbDMDH.In this assay,S-substrate was converted to R-product,which then was oxidized by LbDMDH.The coenzyme NAD+was reduced to NADH during the process accompanying the increase of the absorbance at 340 nm.Therefore,the rate of absorbance increase reflected the racemse activity when excessive LbDMDH was added.The optimum assay consisted of 1 mM NAD+,10 mM(S)-mandelic acid,100 mU LbDMDH and a certain amount of racemase at pH 7.8.Moreover,the procedure was validated with the wild type ArMR and was easily amendable to other substrates as a high-throughput screening approach.