Modification Of Nitrile Hydrase Activity And Thermal Stability Based On Semi-rational Design And Rational Design

Nitrile hydratase?NHase;EC:4.2.1.84?is a kind of metal enzyme that catalyzes nitriles to amides.Amides are widely used as chemical raw materials or pharmaceutical intermediates.Due to the advantages of simple operation,high catalytic efficiency and no by-products,the biological synthesis method has replaced the chemical synthesis method become the main method for the production of amides in industry.However,most industrial NHases have low activity and low thermal stability,which greatly limits the further application of NHase in industry.Based on this,this study modified PtNHase from Pseudonocardia thermophila JCM3095 to improve its catalytic performance.The main research results are as follows:?1?Improve PtNHase activity based on semi-rational design.Part of amino acid residues located in the substrate-binding pocket of PtNHase were mutated,and the enzyme activity of M46K was significantly improved.Its activity to nicotinonitrile was 5.79 times of the wild-type NHase.The mutant F168Y was screened by sequence alignment,it's activity to nicotinonitrile was 2.16 times of wild-type NHase.By constructing the combination mutant M46K-F168Y,the specific enzyme activity of NHase to nicotinonitrile was increased to 574.02±17.5 U·mg^-1,which was 7.45 times of the wild type NHase.?2?Improve the thermal stability of NHase based on rational design.With PtNHase as the starting protein and assisted by Fireprot,a mutant M10 with significantly improved thermal stability was successfully obtained,the half-life of M10 at 62?is 150 minutes which is 7.6times of wild-type NHase,the specific enzyme activity of M10 was also increased from77.05±1.8 U·mg^-1 to 168.8±5.27 U·mg^-1.?3?In order to further improve the catalytic performance of PtNHase,the mutant M46K-F168Y-M10 was constructed based on the mutant obtained above.Compared with wild-type NHase,the specific enzyme activity and thermal stability of the mutant were significantly improved.The half-life of the mutant was 100 minutes at 62?and 5.56 times of the wild type NHase.The specific enzyme activity was 305.37±3.22 U·mg^-1,which is 3.96 times that of wild-type NHase.Meanwhile,the product tolerance of mutant was 15%higher than that of wild-type NHase.