烟酰胺磷酸核糖转移酶（Nicotinamide phosphate ribose transferase，Nampt）是生物酶法合成烟酰胺单核苷酸（Nicotinamide ribotide，NMN)中重要的酶，催化烟酰胺（Nicotinamide，NAM）和磷酸核糖焦磷酸（Phosphoribosyl pyrophosphate，PRPP）合成NMN。本研究将来源Meiothermus ruber的Nampt在大肠杆菌系统进行胞内表达，表达产物经纯化后进行酶学性质分析，并进一步将其用于催化合成NMN。将重组菌株在16 ℃低温下进行摇瓶水平诱导21 h，收集发酵菌体并进行超声破碎，破碎后上清利用Ni-NTA螯合亲和层析的方法进行纯化，SDS-PAGE结果显示表达与纯化后的产物大小约55 ku，与预期的蛋白分子量相符。重组Nampt的最适反应温度为45 ℃，最适pH为6。酶动力学分析显示，该酶对底物NAM催化的Km、Vmax、kcat分别是0.39 μmol/L、3.20 μmol/(mg·min)、1.39 1/s。使用该酶催化生产NMN，通过反应中补加一次底物PRPP，反应10 min产物NMN产量可达30 mg/L。本研究成功表达一个酶学性质和热稳定性优良的Nampt，并将其应用在单酶催化生产NMN时有较高的产量和生产效率，为生物酶法合成NMN的应用研究奠定了基础。

Nicotinamide phosphate ribose transferase (Nampt) is an important enzyme in the bioenzymatic synthesis of nicotinamide ribotide (NMN), and is used to catalyze the synthesis of NMN from nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP). In this study, Nampt from Meiothermus ruber was expressed intracellularly in the Escherichia coli expression system, and the expressed product was purified for the examinations of enzymatic properties and catalysis for the synthesis of NMN. Recombinant strains were induced in shake flasks at a low temperature (16 ℃) for 21 h. The fermented cells were collected and disrupted ultrasonically. After the disruption, the supernatant was purified by Ni-NTA chelation affinity chromatography. SDS-PAGE results showed that the size of the expressed and purified product was about 55 ku (which was consistent with the expected protein molecular weight). The optimal reaction temperature of the recombinant Nampt was 45 ℃, with the optimal pH as 6. Enzyme kinetic analysis showed that the Km, Vmax, and kcat of the enzyme on the substrate NAM were 0.39 μmol/L, 3.20 μmol / (mg·min), and 1.39 1/s, respectively. The enzyme was used to catalyze the production of NMN, and the product NMN yield could reach 30 mg/L with an addition of the substrate PRPP within the 10 min reaction. In this study, a Nampt with excellent enzymatic properties and thermal stability was successfully expressed, and applied to a single enzyme-catalyzed production of NMN with a higher yield and production efficiency. This research has laid the foundation for the application of bioenzymatic synthesis of NMN.

广东省重点领域研发计划项目（2019B020210001）