Directed Evolution Of Substrate Selectivity For D-mandelate Dehydrogenase

Halogenated mandelate and its derivatives are important scaffolds in chemical industry.Because of their good permeability and stability,they are widely used in organic synthesis,especially in the industrial production of pharmaceutical intermediates.In recent years,enantiomerically pure L-o-chlorophenylglycine,as the key synthesis block of clopidogrel,has attracted extensive attention and research.D-mandelic acid dehydrogenase and mandelic acid racemase play a key role in the biosynthesis of L-o-chlorophenylglycine,However,the existing D-mandelic acid dehydrogenase and mandelic acid racemase have relatively low catalytic activity for D-o-chloromandelic acid,which largely restricts the development of L-o-chlorophenylglycine biosynthesis.Therefore,in order to eliminate the inhibitory effect of the ortho-chloro substitution of o-chloromandelate on enzyme activity and improve the biocatalytic performance of o-chloromandelate,we engineered D-mandelate dehydrogenase with the help of protein engineering technology.to enhance the catalytic performance for halogenated substrates.The main contents and results of this study are as follows:（1）Lb DMDH,a novel D-mandelate dehydrogenase extracted from Lactobacillus brevis,was used as the research object.In order to improve its catalytic activity for halogenated substrates,a D-mandelate dehydrogenase mutant Lb DMDH^N253Swith higher catalytic activity for D-o-chloromandelic acid was successfully obtained by directional evolution,and its catalytic activity was about 29 times that of Lb DMDH.In addition,the optimal temperature of Lb DMDH^N253Sis 50℃,which is closer to the optimal growth temperature of E.coli,indicating that it may be more favorable in whole cell catalysis with living cells as catalysts.Meanwhile,the K_catvalue of Lb DMDH^N253Sfor D-o-chloromandelic acid was 2.08 s^-1,which was significantly higher than that of Lb DMDH(0.09 s^-1),indicating that Lb DMDH^N253Shad higher catalytic efficiency for D-o-chloromandelic acid.（2）Using the D-mandelic acid dehydrogenase Lb DMDH from Lactobacillus brevis as the probe,a novel D-mandelic acid dehydrogenase Lh DMDH was obtained from Lactobacillus harbinensis by genome mining technology.Its activity against D-mandelic acid is up to 1200 U/mg,which is in the leading position in the reported D-mandelic acid dehydrogenase.Compared with Lb DMDH derived from Lactobacillus brevis,Lh DMDH derived from Lactobacillus harbinin had higher catalytic activity for D-mandelic acid and D-o-chloromandelic acid,which was about4 and 2 times than that of the Lb DMDH respectively.Therefore,in this study,the semi-rational design-assisted directed evolution of D-mandelate dehydrogenase Lh DMDH was carried out.First,two mutant enzymes Lh DMDH^D52Gand Lh DMDH^A59Vwith slightly improved activities were constructed by random mutation.Furthermore,we have determined that the Lb DMDH^N253Smutant has significant activity against D-chloromandelic acid in our previous study and that the 253rd asparagine of Lb DMDH corresponds to the 252nd asparagine of Lh DMDH.Therefore,sites 52,59 and 252 of Lh DMDH were selected as the key sites for further research.Using p ET28a-Lh DMDH as a template,single-point mutation was introduced into the Lh DMDH gene sequence by whole-plasmid PCR method to construct a single-point mutant strain.According to the screening results,some mutants with a larger increase in enzyme activity were selected,and the combined mutants were constructed by the whole plasmid PCR method,in order to achieve the purpose of superimposing the advantages of mutation.After three rounds of laboratory evolution,a D-mandelate dehydrogenase mutant with high catalytic activity,Lh DMDH^A59G-N252G,was successfully obtained,and its activity was about 6 times that of Lh DMDH and about10 times that of Lb DMDH^N253S.Lh DMDH^A59G-N252Gexhibited excellent p H properties,expression levels,specific activity and kinetic parameters compared to wild type.Taking the concept of green development as the guiding ideology,this study aims to break through the key technical bottleneck restricting the production efficiency of clopidogrel intermediate.The successful implementation of this study is expected to reveal the molecular mechanism of the substitution position of D-chloromandelic acid chloride affecting the activity of D-mandelic acid dehydrogenase,broaden the application scope of D-mandelic acid dehydrogenase,provide new ideas for the selective transformation of other enzyme substrates,and have important academic significance for enriching and perfecting the basic theories and methods of enzyme engineering.