Heterologous Expression And Enzymatic Property Study Of A Novel Thermostable Nitrile Hydratase

Nitrile hydratase?NHase,EC 4.2.1.84?is a metalloenzyme found in prokaryotic and eukaryotic organisms that catalyzes the hydration of nitriles to amides and is mainly used in the industry to produce nicotinamide and acrylamide.Amides,as important chemical raw materials and organic synthesis intermediates,are widely used in the production of bulk chemicals and medicines.At present,thermal stability is very important for the industrial applications of nitrile hydratases,but the poor thermal stability of most nitrile hydratases becomes a limitation.With the sequencing of the genomes of a large number of microorganisms,the number of annotated and unresolved nitrile hydratase genes in the Gene Bank is rapidly increasing.Mining new genes and developing new technologies will help promote the“green”production of amide compounds in the industry and make important contributions to environmental protection.In this study,a novel thermostable nitrile hydratase was discovered and it was heterologously overexpressed in E.coli BL21?DE3?.Its enzymatic properties,thermal stability,and the potential for industrial application were studied.The main contents are as follows:?1?A novel nitrile hydratase was found by BLAST in the Genebank of NCBI from the starting point of heat resistance.The strain was Cal.thermarum TA2.A1,of which the optimum growth temperature was 65?70?.After codon optimization of the NHase gene,the expression vector of pET-24a?+?-Cal.t NHase was constructed and then heterologously expressed in E.coli BL21?DE3?.The protein was purified by adding strep-tag at the C-terminus of the?subunit.?2?The specific activity of Cal.t NHase towards 3-cyanopyridine was 395 U·mg^-1 at 30?.The residual activity of the novol enzyme at 60?for 7 h was 57.5%and the half-life at 70?was determined to be 14 min.Cal.t NHase could maintain 52.8%of its activity after 30 min of treatment with 1 M nicotinamide and 53.3%of its residual activity after 30min of treatment with 2 M nicotinamide.In the whole-cell catalysis process,adding 0.4 M nicotinamide powder every 6 min in the early stage was finally selected by optimizing the feeding concentrations and times,and the final concentration of nicotinamide was 495 g·L¹.The nicotinamide yield of Cal.t NHase was 26.9%higher than that of H-NHase from R.rhodochrous J1.?3?It was found that the?His150 of Cal.t NHase was crucial to its thermal stability by comparing the RMSF between Cal.t NHase and Pt NHase.When?His150 replaced any other amino acid,the Cal.t NHase mutants showed poor thermal stabilities.It was found by kinetic simulation that the?His150 interacted with the Trp203 of another?subunit to form the hydrogen bond which stabilized the protein.The?His150 played a key role in the thermal stability of Cal.t NHase in this study.So the?Asn154 of H-NHase of R.rhodochrous J1corresponding to the?His150 of Cal.t NHase was mutanted to the His.The thermal stability and activity of the H-NHase-?N154H mutant were increased and it was useful to the industrial application.