Studies On Regioselectivity And Catalytic Properties Of The Nitrile Hydratase Towards Adiponitrile

Nitrile hydratase (NHase, EC4.2.1.84) is a kind of metal enzyme that catalyzes cyanogroup of nitriles to amide group. The mature NHase is composed of alpha and beta subunit,usually forming α2β2tetrameric molecule. NHase catalyzes mono-cyano substrate, such asnicotinonitrile and acrylonitrile, to produce nicotinamide and acrylamide respectively. As toother nitriles, such as di-nitriles and multi-cyano substrate, the catalytic properties are unclearyet. In this study, the nitrile hydratase from Pseudomonas putida NRRL-18668(PpNHase)was set to the research model. The recombinant plasmid pET24a-bap was expressed in E.coliBL21(DE3). After ultro-sonication, the supernatant was rudely separated by ammoniumsulfate precipitation (40%~75%). Next, the collected proportions were dialysis prior to beingsubjected to Hitrap Q HP anion exchange chromatography, Superdex G200gelchromatography. By measuring the specific activity of the purified enzyme obtained beforeand after purification, the purification fold was determined to11.6. High performance liquidchromatography (HPLC) and UPLC/Q-TOF MS were done to measure catalytic capability ofadiponitrile by PpNHase. The results showed that the optimum reaction temperature was35°C, the optimum reaction pH was7.4. Notably, the wild type PpNHase catalysed onecyano group of adiponitrile into amide and generated5-cyanopentanamide, suggesting thatthe PpNHase exhibits catalyzing regioselectivity.To further investigate the molecular basis of regioselectivity, seventeen single-pointmutants were built by using wild-type PpNHase gene as the template. Each mutant wasexpressed and purified. catalytic products of adiponitrile by each mutant were detected withHPLC, the data showed that Leu37Phe mutant or Ala97truncated mutant was completely ableto catalyze the adiponitrile to adipamide. Substrate docking comparison by homologymodeling structure showed that Leu37Phe had the capacity of altering the size of substrateaccess channel, resulting in whether producing intermediate5-cyanopentanamide oradipamide. These data also showed that Ala97at β chain, which located in the substrateaccess of adjacent αβ complex channel inlet, indirectly effected on importation of5-cyanopentanamide to the active center, eventually producing regioselectivity catalysis.Furthermore, this study also explored the catalytic capability of nitrile hydratase fortri-cyano nitrile substrate hexane-1,3,6-tricarbonitrile by using HPLC and UPLC/Q-TOF MStechniques. We found that nitrile hydratase was able to catalyze any one cyano group, twocyano group or all three cyano group of hexane-1,3,6-tricarbonitrile respectively, generatingcorresponding mono-amide, di-amides and tri-amides.