Molecular Modification And Application Of Nitrilase

Nitrilases(EC 3.5.5.1)are important industrial enzymes used to convert nitriles directly into corresponding carboxylic acids and ammonia.Nitrilases have been employed to convert nitriles in a single step with mild conditions,fast reaction and less amounts of byproducts,which is preferred to chemical methods.Studies on nitrilases have been widely carried out in terms of their occurrences,catalytic mechanisms,applicabilities,cloning of the nitrilase genes from different origins,and directed evolution of nitrilases.Recently,screening new nitrilases,exploring their new applications and molecular modification of nitrilases are becoming hot issues.In our previous work,a strain of Alcaligenes faecails ZJUTB10 with high nitrilase activity in converting racemic mandelonitrile to(R)-(-)-mandelic acid was isolated and characterized.The nitrilase specific activity reached 350.8 U/g and highest production rate were 9.3 mmol h-'g-1.Then the fragment with the length of 1068 bp encoding the A.faecalis ZJUTB10 nitrilase was obtained.Due to its high enzymatic activity and tolerance to the high concentration of the products,this strain seems to possess some potential applications for the industry to produce of carboxylic acids from nitriles.In this study,the nitrilase was molecular modified at the gene level,which has been conserved in the laboratory.Due to the secondary structure of the nitrilase,substrate and the nitrilase docking study,we select the three loci in the three a-helix(L175,A197,V305),a locus in one of the?-sheet(A2 84),which are located after Cys-163.And using the software HotSpot Wizard to identify the point that plays an important role in enzyme activity and substrate specificity,a point of Q196 was selected.Then five pairs of primers were designed.The recombinant plasmid pET28b-NIT was used as the template for single-point or complex point saturation mutagenesis.Approximately 6300 bp mutant fragment of the restructuring plasmid library was accessed;rebuild the E.coli recombinant nitrilase mutants E.coli BL21(DE3)/pET28b-NITmut.After the high-throughput and liquid chromatography screening,a number of mutants with increased activity,improved stability,substrate and product tolerance were obtained.Enzymatic properties of the mutated nitrilases indicate that,catalytic activity of the nitrilases AA284V and AQA284I was increased in the low pH conditions,and their pH stability is significantly improved,too.The optimum temperature of mutated nitrilases is almost the same,about 40 °C.But the thermal stability increased slightly.Homology modeling and molecular docking study can predict spatial structure and catalytic mechanism of nitrilase.The results show that the diameter of AA284V catalytic tunnel is the maximum,equal to 2.06 A,and tunnel volume 15.0 A3 is close to that of AQ196S and AQA284I.Finally,the best mutant(AA284V)was selected for the study of mandelic acid production.Results show that under the conditions of substrate mandelonitrile 6%,the yield of mandelic acid was increased from 72%to 80%.Substrate feeding experiments were conducted with Dual-channel pH-titrator,in the conditions of constant pH.Conversion rate of mandelonitrile(6%)was more than 95%,and the yield of mandelic acid was reached 90%.