Engineering Of Nitrilase And Its Application In Biosynthesis Of(R)-Mandelic Acid

Enantiomerically pure 2-hydroxyarylacetic acids are valuable intermediates and building blocks for the synthesis of biologically active compounds and target molecules.For instance,R-mandelic acid is the important fine chemical intermediates and chiral prodrug with broad uses in pharmaceutical and chemical industry.As nitrilases are important industrial enzymes that convert mandelonitrile directly into the R-mandelic acid,the nitrilase-mediated reaction has exciting potential due to the cheap starting material and the 100%theoretical yield.In this study,the molecular docking experiment of mandelonitrile into the nitA active site.Residues?Thr132,Ser190?were mutated separately by site-directed mutagenesis.Site 190 was demonstrated to be the critical position,which has a greater influence on arylacetonitrilase activity and enantioselectivity.The size of the side-chain of the substituted amino acid affected the arylacetonitrilase catalytic performance significantly.The replacement of the residue serine at position 190 with glycine markedly increase its activity and enantioselectivity towards mandelonitrile and?o,m,p?-chlorommandelonitrile,whereas,replacing it with leucine abolished its activity.The best mutant,exhibited 3-fold higher specific activity towards mandelonitrile compared with that of wild-type nitA.The enantioselectivity was improved from 94%to>99%.In order to elucidate the reason for the increase in the activity from a biochemical perspective,the steady-state kinetic parameters of wild-type nitA and mutant enzymes for the hydrolysis of mandelonitrile and?o,m,p?-chloromandelonitrile were determined.Enzymatic properties indicated that the optimum temperature and pH of mutant was 50°C and pH 7.5,respectively.The best mutant was found to have the highest substrate-binding affinity?K_m,0.76 mM?.The catalytic efficiency(k_cat/K_m)of mutant is 1043.67 min^-1 mM^-1,which is 7.5-fold higher than that of wild-type nitA.The enantioselectivity?E?of variant was improved from145.12 to>200,which shows great potential in industrial application.To obtain a better understanding of the differences observed in activity and enantioselectivity towards the substrates between the wild-type nitA and,the R-and S-isomers of the substrates were modeled into the active site of wild-type nitA and,respectively.We also analyzed the change of catalytic tunnel between the wild-type nitA and.The results indicated that the residue at position 190 has a great impact on the spatial structure of catalytic center.The diameter and volume of the catalytic tunnel for were 1.48 and 13?3 compared with 0.65 and 25.0?3for the wild-type.The larger catalytic tunnel and smaller tunnel volume can improve the nitrilase to hold and selectively catalyze the substrates.To verify the improvements offered by the mutant in practical applications,biocatalytic hydrolysis of mandelonitrile was conducted with a substrate?mandelonitrile?loading of 100 mM.The reaction catalyzed by the mutant reached 100%conversion with above 99%e.e._p after 30 min,whereas the conversion in the case of the wild-type nitA was only 60%with 94%e.e._p after 30 min.The reaction rate of-mediated hydrolysis of mandelonitrile reached 200 mmol/L/h.This is the highest reaction rate among all the existing nitrilases aimed at producing?R?-mandelic acid from mandelonitrile.To relieve the substrate inhibition and improve the productivity,a biphasic system of toluene-water?2:8,v/v?was adopted,in which a maximum of catalytic 1M mandelonitrile converting to generate 841.24 mM of?R?-mandelic acid and e.e._p>99%within 11.5h.The engineered mutant is a promising biocatalyst for practical applications in synthesizing?R?-mandelic acid.