烟酰胺单核苷酸（NMN）是具有重要医用价值的NAD+前体，通过信号肽PelB实现大肠杆菌BL21(DE3)分泌表达催化NMN合成的烟酰胺磷酸核糖转移酶（Sfnampt）、磷酸核糖焦磷酸激酶（Tkprs）和核糖激酶（Erbks）。对三酶的酶学特性分析显示，Sfnampt、Tkprs和Erbks的最适反应温度分别为45、50和37 ℃，最适pH值分别为7.5、8.5和5.5。热稳定性分析发现，Sfnampt在35 ℃热稳定性良好而在40~55 ℃较差；Tkprs在40~60 ℃热稳定性良好；Erbks在25~40 ℃热稳定性良好而在45 ℃较差。酶动力学分析可知，Sfnampt、Tkprs和Erbks的Vmax分别为0.65 μmol/(L•min)、2.37 μmol/(L•min)、12.58 μmol/(L•min)，Km分别为2.87 μmol/L、25.48 μmol/L和74.13 μmol/L，Tkprs、Erbks与已表征的Prs、Rbks相比底物亲和力好。将分泌表达的三酶用于催化合成NMN，温度37 ℃、pH值8.0为较优催化条件。底物ATP以及酶Sfnampt在反应体系中为关键因素，经底物和酶配比优化后，使用三酶经过7 h催化可获得5.50 μmol/L的NMN。该研究在大肠杆菌系统成功实现了NMN合成途径酶的分泌表达，为NMN合成提供了新思路。

Nicotinamide mononucleotide (NMN) is a precursor of NAD+ with important medical value. Escherichia coli BL21(DE3) expresses and secretes the enzymes nicotinamide phosphoribosyltransferase (Sfnampt), phosphoribosyl pyrophosphate kinase (Tkprs), and ribokinase (Erbks), which catalyze the synthesis of NMN through the signal peptide PelB. The enzymatic characteristics of three enzymes were analyzed, and the results showed that the optimum reaction temperatures of Sfnampt, Tkprs, and Erbks were 45 ℃, 50 ℃, and 37 ℃ respectively, and the optimum pH values were 7.5, 8.5, and 5.5 respectively. In terms of thermal stability, Sfnampt was found to have good thermal stability at 35 ℃ and poor stability at 40~55 ℃. The thermal stability of Tkprs was optimized at 40~60 ℃, whereas that of Erbks was best at 25~40 ℃ and poor at 45 ℃. The results of the enzyme kinetic analysis showed that the Vmax values of Sfnampt, Tkprs, and Erbks were 0.65, 2.37, and 12.58 μmol/(L•min), respectively, and Km values were 2.87, 25.48, and 74.13 μmol/L, respectively. Compared with the characterized Prs and Rbks, Tkprs and Erbks had better substrate affinity. The three enzymes were used for the catalytic synthesis of NMN, and the optimum catalytic conditions were 37 ℃ and pH 8.0. The substrate ATP and enzyme Sfnampt were the key factors in the reaction system. After optimizing the ratio of substrate and enzyme, 5.5 μmol/L of NMN could be obtained after 7 h of catalysis by the three enzymes. In this study, the enzymes for the pathway of NMN synthesis were determined to be successfully expressed and secreted in E. coli, which indicates a new direction for the synthesis of NMN.

国家重点研发计划项目（2018YFA0901700）