腺苷酸环化酶（Adenylate Cyclase，AC）对酶法合成环单磷酸腺苷（Cyclic Adenosine Monophosphate，cAMP）至关重要，它催化三磷酸腺苷（Adenosine Triphosphate，ATP）生成cAMP和焦磷酸（PPi）。本研究将Thermomonospora echinospora来源的AC（TeAC）在大肠杆菌中进行异源表达，经过亲和层析纯化蛋白后进行酶学性质的分析，并进一步将其用于cAMP的催化合成。在16 ℃下诱导重组大肠杆菌表达TeAC后，利用Ni柱亲和层析纯化TeAC，经过SDS-PAGE分析表明目的蛋白条带为40 ku，与预期蛋白大小一致。重组TeAC酶的最适温度为50 ℃，最适pH 值为10.5。经酶动力学分析，测得该酶对底物ATP催化的动力学参数米氏常数（Km）=115.1 mmol/L，最大反应速度（Vmax）=64.52 μmol/(mg•min)，催化常数（Kcat）=8.13s-1。用TeAC催化底物ATP反应11 h后cAMP产量可达19.1 g/L。该研究成功表达了一种具有高效催化性能的TeAC，并将其应用于cAMP的催化合成时有较高的产量和生产效率，为酶法合成cAMP 的应用研究奠定了基础。

Adenylate cyclase (AC) is essential for the enzymatic synthesis of cyclic adenosine monophosphate (cAMP). In fact, AC catalyzes the synthesis of cAMP and pyrophosphates (PPi) by adenosine triphosphate (ATP). In this study, Thermomonospora echinospora-derived AC (TeAC) was heterologously expressed in Escherichia coli. Following purification of the protein via affinity chromatography, its enzymatic properties were analyzed. Thereafter, the protein was utilized for the catalytic synthesis of cAMP. After inducing the expression of recombinant TeAC in E. coli at 16 ℃, TeAC was purified via affinity chromatography on a Ni column. Based on SDS-PAGE, the band of the protein of interest corresponded to 40 ku, which aligns with the expected protein size. The optimal temperature and pH of recombinant TeAC enzyme were 50 ℃ and 10.5, respectively. Enzyme kinetic analysis revealed Km, Vmax, and Kcat values of 115.1 mmol/L, 64.52 μmol/(mg•min), and 8.13 s-1, respectively, for enzymatic catalysis using the ATP substrate. After 11 h of the reaction catalyzed by TeAC, the yield of cAMP was 19.1 g/L. Overall, TeAC with efficient catalytic performance was successfully expressed, and its application in the catalytic synthesis of cAMP led to increased yield and production efficiency. This study lays the foundation for the enzymatic synthesis of cAMP.

国家重点研发计划项目（2018YFA0901700）