Study On The Biosynthesis Of Nicotinamide Adenine Dinucleotide

Nicotinamide adenine dinucleotide(NAD+)is an important coenzyme in life,and plays an important role in life processes such as redox,gene expression,energy metabolism,regulating cell life cycle and disease.The synthesis methods of NAD+are divided into chemical and biological types.Compared with chemical methods,the preparation of NAD+by biocatalysis has the advantages of simple process conditions,green environmental protection,and short time consumption.In this paper,nicotinamide mononucleotide adenosine transferase(NMNAT)and nicotinamide riboside kinase(NRK)can be used as biocatalysts to establish a NAD+catalytic reaction system.NRK can catalyze the reaction between NR and ATP to get NMN,NMNAT can continue to catalyze the reaction between NMN and ATP to get NAD+.Using bioinformatics methods,five NMNAT encoding genes were screened from the NCBI database,and the target gene N-cva that could efficiently express catalytic activity in E.coli was identified,thereby obtained a nicotinamide mononucleotide adenosine transferase which can be used for the preparation of NAD+.The optimal fermentation conditions of engineering bacteria N-cva were explored,and the optimal fermentation conditions of N-cva were determined as follows:when the LB medium OD600=0.7,the inducing agent IPTG with a final concentration of 0.1 mM was added,the induction temperature was 32?,and the induction duration was 6h.A high-density fermentation test was carried out in a 5L fermentor,and finally about 180g of wet cells were obtained.The enzymatic properties of the engineering bacteria N-cva were explored and found to have good thermal stability.It could retain about 80%of the activity under the optimal reaction temperature under a high temperature environment of 90?.After treatment at 70? for 1h,it still retained about 60%of its activity,the activity under neutral and alkaline conditions was higher than acidic conditions.The catalytic process of N-cva was studied,and the optimal reaction conditions were determined as:reaction temperature was 40-50?,reaction pH=7.0,the reaction buffer was 50mM imidazole-phosphate buffer,and enzyme-to-bottom ratio 1.3:1.The catalytic reaction was set according to the optimal reaction conditions,and the yield of NAD+could reach more than 92%.NRK was efficiently expressed in E.coli,and the optimal fermentation conditions of NRK were determined as follows?when the LB medium OD600=0.7,the inducer IPTG with a final concentration of 0.1 mM was added,the induction temperature was 28?,and the induction duration was 12h.The catalytic process of NRK was explored.The optimal catalytic process conditions were:the reaction temperature was 40?,the reaction pH=5.0,and the reaction enzyme bottom ratio was 0.8:1.The catalytic reaction was set according to the optimized reaction conditions,and the yield could reach more than 90%.The reaction process in which NRK and N-cva catalyze the reaction of NR and ATP to form NAD+ in the same system were constructed.Under the respective optimal reaction conditions,the final yield of the catalytic reaction of NRK and N-cva could reach 80%.