Biological Enzymatic Synthesis Of Nicotinamide Mononucleotide

Nicotinamide mononucleotide(NMN)is an important precursor for the synthesis of NAD+ in the human body.It is converted into NAD+ to exert its physiological functions and is closely related to various diseases.Among them,NMN is used to treat aging diseases and delay aging,which is one of the most popular areas of its research.NMN has been gradually used in health foods and medical drugs for the prevention and treatment of metabolic diseases,cardiovascular and cerebrovascular diseases,aging diseases,etc.However,due to production problems,products with NMN as the main component are currently very expensive.In the method of producing NMN,compared with the chemical method for synthesizing NMN,the biological method is the most environmentally friendly and pollutionfree production method because it has no residual organic solvents and no chiral problems.However,there are still some limitations,such as the lack of enzyme catalysts with high catalytic efficiency,and high production costs caused by the reaction substrate.The purpose of this study was to find a Nicotinamide phosphoribosyl transferase(Nampt,EC 2.4.2.12)suitable for the production of NMN through a enzyme molecular screening method based on bioinformatics.The enzyme is a catalyst for nicotinamide(NAM)and phosphoribosylpyrophosphate(PRPP)directly synthesizes the enzyme of NMN(nicotinamide mononucleotide).Based on this,the enzyme is initially applied to the production of NMN by single enzyme and multiple enzymes.The specific research results are as follows:(1)Mining Nampt by molecular screening method based on bioinformaticsSo far,there are more than 600 records of nicotinamide phosphoribosyltransferase in KEGG database,only those from Homo sapiens,Mus musculus and Rattus norvegicus have been characterized,which is difficult to meet the needs of NMN synthesis.In this study,the amino acid sequence of Nampt from Meiothermus ruber and Methanobcterium sp.Pta U1.Bin097 was used,Nampt is filtered by Blastp in NCBI database,the Discovery Studio software and molecular docking method was used,by simulating the binding of Nampt from different sources with the substrate small molecule NAM in the binding pocket,a scoring system with a high-CDOCKER_ENERGY score was selected as the Nampt of the docking result.Candidate enzyme catalyst.Among mammals,the highest-CDOCKER_ENERGY value is Nampt from Homo sapiens(h Nampt),the highest-CDOCKER_ENERGY value from bacteria is Nampt from Comamonadaceae bacterium(c Nampt)and Meiothermus ruber(m Nampt).Nampt from these three sources was used as candidate enzyme.(2)Expression and analysis of enzymatic properties of Nicotinamide phosphate ribose transferaseIn this study,the three Nampt genes were expressed in Escherichia coli,and the Nampt protein was purified.The enzymatic properties of Nampt from three sources were analyzed.With Nam as substrate,the km of h Nampt were 0.12 ?mol / L,Vmax 0.11 ?mol /(min · mg),and kcat 0.05 1/s.The Km,Vmax and kcat of m Nampt were 0.39 ?mol / L,3.20 ?mol /(min · mg)and 1.39 1/s,respectively.The Km,Vmax and kcat of c Nampt were 0.14 ?mol / L,3.10 ?mol /(min · mg)and 1.38 1/s respectively.Compared with the characterized Nampt,the kcat / Km of m Nampt and c Nampt expressed in this paper is much higher than that of Nampt from other sources,which is more suitable for industrial enzymatic production of NMN.Among them,kcat / Km of c Nampt is 9.86 × 106 1 / s,which is the highest among Nampts reported so far.The kinetic parameters measured were discussed with the results of molecular docking in the above enzyme molecular screening method.The order of the-CDOCKER_ENERGY size of the Nampt molecular docking results from the three sources is consistent with the size of the enzyme kcat / Km determined by heterologous expression,and the fitting degree was high.This shows that the molecular screening method in the early stage of this study has a good effect on the screening of Nampt with good catalytic properties,which verifies the usability of this screening method.(3)Catalytic production of NMN with single and multiple enzymescNampt was used to catalyze the synthesis of NMN by single enzyme.The results showed that in the synthesis of NMN by single enzyme method,the yield of NMN was 34 mg / L at 10 min and the conversion efficiency was 204 mg / L · h when PRPP was added once.Based on the analysis of literature,the Ribophosphate pyrophosphate kinase(Prs)Km of H.sapiens(h Prs)is much higher than that of other species,and the Prs specific activity of Pyrobeauum calidifontis(pPrs)is is the highest.The two Prs were expressed in E.coli,and the Prs protein was purified.Among them,h Prs was successfully expressed in soluble form,while pPrs was not expressed in soluble form.NMN was synthesized by the combination of h Prs and c Nampt.The yield of NMN was 29 mg / L at 180 min and the conversion efficiency was 9.67 mg /(L · h).Based on the analysis of literature,the Ribokinase(Rbks)with better enzymology property was analyzed.The substrate affinity of Rbks from E.coli(e Rbks)was the best among the prokaryotic Ribokinase,and the substrate affinity of Ribokinase from H.sapiens(h Rbks)is the best one among the mammalian ribokinase,and the kcat / Km value is the highest of Rbks.The two Rbks were expressed and purified in in E.coli,and the Rbks protein was purified.NMN was synthesized by e Rbks,h Prs and c Nampt.The yield of NMN was 24 mg / L at 240 min and the conversion efficiency was 6 mg /(L · h).NMN was synthesized by h Rbks,h Prs and c Nampt.The yield of NMN was 24 mg / L at 180 min and the conversion efficiency was 8 mg /(L · h).From single enzyme to multiple enzymes to synthesize NMN,with the increase in the number of enzymes participating in the reaction,although the yield has decreased,the double enzyme method is reduced to 85% of the single enzyme method,and the three enzyme method is reduced to 71% of the single enzyme method.However,the total cost of substrates has also been greatly reduced,of which the double enzyme method is reduced to 1% of the single enzyme method,and the three enzyme method is reduced to 0.4% of the single enzyme method.