Study On The Catalytic Properties Of Recombinant Nitrile Hydratase From Rhodococcus Erythropolis CCM2595

Background: Amides are vital fine chemical raw materials and pharmaceutical intermediates.Nitrile hydratase is a metal enzyme capable of catalyzing the hydration of nitrile compounds to amide compounds which has high industrial application value.Compared with the traditional chemical synthesis methods,biocatalysis methods for producing amide compounds have the advantages of mild reaction conditions,strong specificity and high catalytic efficiency.However,at present,the methods for obtaining wild-produced enzyme strains from natural soil sampling and screening are limited by chance,low success rate,and poor enzyme activity.The genetically engineered bacteria that efficiently expresses nitrile hydratase provides a new strategy.The main advantages of gene cloning are clear biological genetic background,strong adaptability and short fermentation cycle.These make it conducive to large-scale cultivation and industrial production,simultaneously in line with the development direction of economical and environmentally friendly green chemicals.Purpose: To explore the relative performance of nitrile hydratase from Rhodococcus erythropolis CCM2595,with a view to provide a theoretical basis for the industrial production of amides.Methods:(1)Design of plasmids: heterogeneous expression of nitrile hydratase in E.coli through genetic engineering technology;(2)Study on substrate specificity of nitrile hydratase: use resting engineering bacteria cells to react with different nitrile substrates(acrylonitrile,malononitrile,niconitrile,adiponitrile,terephthalonitrile,benzonitrile),and use gas liquid chromatography to detect the reaction results;(3)Exploration of factors affecting the resting cells reaction: explore the effect on enzyme activity from the aspects of temperature stability,pH stability,optimal temperature,optimal pH,and substrate tolerance;(4)Purification of nitrile hydratase: add His-tags to improve the plasmid design and select affinity chromatography to obtain purified enzymes.Results:(1)Successfully achieved soluble heterologous expression of nitrile hydratase in E.coli;(2)The recombinant bacteria had a weak catalytic effect on mononitrile,while it had good catalytic activity on dinitrile and showed a very excellent regional selectivity.It can completely catalyze 20 mM/L adiponitrile in 10 minutes.The selectivity to 5-cyanovaleramide was 95 %,and the enzyme activity can reach 635 U/g(DCW);(3)The optimal temperature of the recombinant bacteria when catalyzing the adiponitrile reaction was 35 ℃,the optimal pH was 7.4,and it had good stability under room temperature and pH neutral conditions.It can tolerate 200 mM/L adiponitrile and completely converted the substrate within 5 hours;(4)By adding the His-tag sequence after the α-subunit,a new plasmid was constructed and the purification operation was completed.The molecular weight of the purified enzyme single subunit was 27 KDa,and the specific enzyme activity catalyzing adiponitrile was 2917 U/g.Conclusion: Nitrile hydratase from Rhodococcus erythropolis CCM2595 has been functionally and stably expressed in E.coli.This nitrile hydratase had a wide spectrum of substrates and a significant catalytic activity for dinitrile.Under normal temperature and neutral conditions,it can efficiently catalyze the formation of 5-cyanovaleramide from adiponitrile in a short period of time with good substrate tolerance.The results of this experiment were expected to provide new ideas and solutions for the industrial production of 5-cyanovaleramide and also contribute to the development of green chemistry.