Catalytic Properties Of The Purified Nitrile Hydratase From Rhodococcus Erythropolis CCM2595 And Its Encapsulation

Nitrile hydratase（NHase）is a metallase used to catalyze the formation of amides by water synthesis of cyanyl compounds containing carbon and nitrogen triple bond organic functional groups.Because the nitrile hydratase product specificity is high,can be operated under normal temperature pressure,less byproducts,reduce pollution and other advantages,more suitable for the development direction of green chemistry.At present,biocatalysis technology has gradually replaced traditional chemical methods in the field of chemical industry,agriculture and medicine.However,the free nitrile hydratase has many defects,such as poor stability,difficulty in preservation and easy deactivation,during the catalytic reaction and its preservation.Therefore,to explore the catalytic performance of nitrile hydratase and make immobilized nitrile hydratase nanoscale biocatcatalyst through the immobilization of the enzyme can not only solve many problems existing in the free enzyme,but also achieve continuous industrial production,which has great economic value and sustainable development significance to China.A nitrile hydrase（ReNHase）from Rhodococcus erythropolis CCM2595 was found in the previous work of this project.In this study,its enzymological properties and catalytic performance after purification and coating were further explored.The main research results are as follows:（1）The plasmid with histidine tag for purification of ReNHase gene was successfully constructed and heterologously expressed in recombinant Escherichia coli,and ReNHase was purified by AKTA protein separation and purification apparatus.（2）The substrate spectrum of ReNHase was explored by high performance liquid chromatography.It was found that ReNHase showed significant catalytic activity and regional selectivity for dionitrile compounds,especially adipic nitrile.Compared with the whole cell catalysis,the catalytic time of purified ReNHase on the above substrate spectra was significantly shortened,and the conversion of mononitrile compounds was significantly improved.（3）The kinetic constants of the enzymatic reaction of ReNHase to adiponitrile were detected and analyzed.The specific enzyme activity of ReNHase was 63.107 U/mg,which was104 times higher than that of whole cell catalysis.The apparent Km value of reaction with adipic nitrile is 6.6252 mmol/L,indicating that ReNHase has a good affinity with adipic nitrile.The reaction Kcat was 82.77s^-1 and Kcat/Km was 1.249×10⁴(mol^-1*L*s^-1),indicating that ReNHase had a high conversion rate of adipic nitrile.At the same time,the optimum reaction temperature of nitrile hydratase was 35℃,and the optimum reaction p H was 7.4.It was found that the catalytic activity of ReNHase could still maintain more than 70%in the range of p H 5-9,indicating that ReNHase had good acid-base stability.（4）Preparation of zeolite imidazole salt framework ZIF-67 and ReNHase@ZIF-67biocatalyst.ZIF-67 was characterized by various characterization methods（scanning electron microscopy,thermogravimetric analysis,etc.）,and the experimental results showed that ZIF-67 was successfully synthesized.The pore diameter of ZIF-67 is 3.49?and that of ReNHase is 2.65?.The preliminary conclusion is that ReNHase@ZIF-67 can be synthesized by in situ synthesis.At the same time,the catalytic activity of the preliminary synthesis of ReNHase@ZIF-67 was determined by using the catalytic reaction.The experimental results showed that,compared with the purified ReNHase,Na Cl still maintained a certain catalytic activity,but the activity was greatly reduced.The reasons were speculated as follows:（1）Renhase is an iron-ion metallase,which may combine the metal active center with Ni²⁺-NTA resin in the purification process,resulting in partial enzyme activity loss.（2）Although polymer-ZIF hybrid can enhance the stability of ZIF under acidic conditions,it will also cause partial enzyme inactivation because imidazole can chelate with metal.CONCLUSIONS:The heterologous expression of nitrile hydratase（from Rhodococcus erythropolis CCM2595）was realized.ReNHase has a wide substrate spectrum,which can catalyze different nitrile compounds under neutral conditions at room temperature.Compared with mononitrile compounds,ReNHase has significant conversion rate,substrate affinity and regional selectivity for dionitrile（especially adiponitrile）.Compared with whole-cell catalysis,the catalytic time of ReNHase with substrate spectrum is significantly shorter.The conversion rate of mononitrile compounds was significantly improved.Although the activity of nitrile hydratase catalyst ReNHase@ZIF-67 decreased to a certain extent,it may be due to（1）ReNHase is an iron ion type metallase,and the metal active center may combine with Ni²⁺-NTA resin in the purification process,resulting in partial enzyme activity loss.（2）Although polymer-ZIF hybridization can enhance the stability of ZIF under acidic conditions,imidazole can chelate with metal,which will also cause partial inactivation of our enzyme.It is hoped to provide some theoretical basis for the immobilization of ReNHase.