Engineering Of Nitrilase And Its Application In Biosynthesis Of (R)-chloromandelic 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?-2-chloromandelic acid??R?-2a?is emerging as the preferred chiral intermediate for the industrial synthesis of the anti-thrombotic agent?S?-clopidogrel,one of the best-selling drugs for heart attack and stroke treatment.The nitrilase-mediated resolution has exciting potential for synthesizing?R?-2-hydroxyarylacetic acids from nitriles due to the cheap starting material and the 100%theoretical yield.In this study,we design several nitrilases with superior activity and enantioselectivitytowardsstericallyhinderedsubstrate2-chloromandelonitrile?rac-1a?by protein engineering for efficient production of?R?-2a with the wild-type 2A6 nitrilase?nitA?.The molecular docking experiment of?R?-1a into the nitA active site to identify active site residues that are close to the substrate.These docking experiment identify four positions?52,132,189 and 190?in the nitA active site that might be suitable targets for engineering the activity and enantioselectivity of nitA towards rac-1a.These positions were randomized by gene site-saturation mutagenesis?GSSM?and four single-site libraries were created.Three single mutants at positions 132and 189 affected nitA activity and enantioselectivity towards substrate rac-1a.Then,combinatory saturation mutagenesis libraries at site 132and 189 were constructed.The best mutant?T132A/F189T?showed significantly higher activity and enantioselectivity towards substrate rac-1a with a 4.69-fold higher specific activity?7.39 U/mg?and the enantioselectivity?E?>200.Enzymatic properties of the mutant T132A/F189T indicated that the optimum temperature and pH of mutant T132A/F189T was 50�C and pH7.5,respectively.Besidese,enzyme was stable at 30�C,40�C and pH 7.5,pH 8.5.The double mutant T132A/F189T was found to have the highest V_max(8.27?mol mg^-1 min^-1)and stronger substrate-binding affinity?K_m,2.15 mM?.The catalytic efficiency(k_cat/K_m)of mutant T132A/F189T is150.66 min^-1 mM^-1,which is 7.26-fold higher than that of wild-type nitA.The enantioselectivity?E?of T132A/F189T variant was improved from17.34 to>200.In order to illustrate the differents in the activity and enantioselectivity between the wide-type nitA and the best mutant T132A/F189T,we modeled the R-and S-enantiomers of rac-1a into the active site of wide-type and mutant nitA.For wide-type nit A,the distances between the SH-group of the catalytic Cys162 with the cyano carbon of the substrate?R?-1a and?S?-1a were 3.79?and 4.09?respectively.For mutant T132A/F189T,the distance between the SH-group of the catalytic Cys162 with the cyano carbon of?R?-1a and?S?-1a were 3.37?and 7.25?respectively.The nucleophilic attack on this cyano carbon becomes easier in virtue of the proper orientation and distance for?R?-1a,oppositely for?S?-1a,so mutant T132A/F189T showed significantly higher activity and enantioselectivity towards substrate rac-1aThe biotransformation of rac-1a using whole cells of mutant T132A/F189T as biocatalysts was investigated.To relieve the substrate inhibition and to improve the productivity,a biphasic system of toluene-water?2:8,v/v?was adopted,in which a maximum of 450 mM of?R?-o-chloromandelic acid was produced with 90%molar yield and ee_p>99%within 3 h.This is the first time that a high space-time yield of?R?-o-chloromandelic acid up to 671.76g�L^-1�d^-1 using a nitrilase-mediated approach is reported.The engineered T132A/F189T mutant is a promising biocatalyst for practical applications in synthesizing?R?-2a.