Research On The Active Nitrilase Aggregates Induced By Self-assembly Peptides

Nitrilase can hydrolyze poisonous mandelonitrile to the R-mandelic acid and ammonia that has attracted extensively attention for the catalytic routes with economical,energy-saving,environmentally friendly and high enantioselectivity.Besides,the product R-mandelic acid has been widely used in fine chemicals,pharmaceutical intermediates and so on.Therefore,nitrilases has great economic value and industrial application prospect.However,the industrial application of nitrilase is limited for the poor stability and sbustrate tolerance of the enzyme.Therefore,it is significant to promote the catalytic characteristic of nitrilase.In this study,coding sequence of three self-aggregate amphipathic short peptides was selected to be attached to the 3' terminus of nitrilase gene individually.Three expression vectors including pET30a-Nit-R18 A,pET30a-Nit-ELK16 and pET30a-Nit-L6 KD,has been constructed successfully.The results of expression showed that fused nitrilases were found mostly in the precipitation as active inclusion bodies.The nirilase aggregations were simpler to be purified and collected just by centrifugation and the recovery of enzyme activity can reach to 90%.The enzyme kinetics parameters were also considered.It was found that the Km of enzyme aggregations were increased and Kcat/Km were decreased to compare with Nit-His,indicating that these short peptides might affect the exposure of the active site and folding of nitrilase.The stability of nitrilase was also investigated with all the nitrilases were stored at 4 oC for one month.It was suggested that the enzyme activity of Nit-His lost more than 50%,and the enzyme aggregations still preserved highly activities.The secondary structures of nitrilase were further studied.The higher proportion of ?-helix was discovered in Nit-R18 A,and the increased ?-sheet was found in Nit-ELK16 and Nit-L6 KD.Besides,the extra experiment of digestion by proteinase K demonstrated that the nitrilase aggregations were different with inclusion bodies.Furthermore,the enzymatic characteristics of nitrilases immobilized by sodium alginate were studied.Although all the immobilized nitrilases had the same optimum reaction temperature and pH,the thermo-stability of nitrilase aggregations were increased remarkably,about 1.5-3.8 fold to Nit-His.The optimum substrate concentration of reaction was also promoted by 125 mmol/L.Finally,the R-mandelic acid was produced by repeated batch using immobilized nitrilases.Nit-R18 A had the best operational stability,retained 70% of its original activity after reused by seven batches and obtained a productivity of 5.8 g mandelic acid l^-1h^-1.