In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(E...In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(EAC)and Bordetella Pertussis(BAC)were expressed in E.coli BL21(DE3)and comparatively analyzed for their activities.As a result,EAC from E.coli MG1655 exhibited a higher activity.However,amount of EAC were obtained in an insoluble form.Therefore,we expressed the first 446 amino acids of EAC(EAC446)to avoid the inclusion body.The effects of induction temperature,incubation time,and incubation p H were further evaluated to improve the expression of EAC446.Subsequently,the reaction process for the production of c AMP with ATP as a starting material was investigated.As none of c AMP was detected in the whole-cell based biocatalytic process,the reaction catalyzed by the crude enzyme was determined for c AMP production.What's more,the reaction temperature,reaction p H,metal ion additives and substrate concentration was optimized,and the maximum c AMP production of 18.45 g·L^-1was achieved with a yield of 95.4%after bioconversion of 6 h.展开更多
基金The National Natural Science Foundation of China(Grant No.21576134,Grant No.21606127,Grant No.21390200,Grant No.21706126)the National Key Research and Development Program of China(Grant No.2016YFA0204300)the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions。
文摘In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(EAC)and Bordetella Pertussis(BAC)were expressed in E.coli BL21(DE3)and comparatively analyzed for their activities.As a result,EAC from E.coli MG1655 exhibited a higher activity.However,amount of EAC were obtained in an insoluble form.Therefore,we expressed the first 446 amino acids of EAC(EAC446)to avoid the inclusion body.The effects of induction temperature,incubation time,and incubation p H were further evaluated to improve the expression of EAC446.Subsequently,the reaction process for the production of c AMP with ATP as a starting material was investigated.As none of c AMP was detected in the whole-cell based biocatalytic process,the reaction catalyzed by the crude enzyme was determined for c AMP production.What's more,the reaction temperature,reaction p H,metal ion additives and substrate concentration was optimized,and the maximum c AMP production of 18.45 g·L^-1was achieved with a yield of 95.4%after bioconversion of 6 h.
文摘基于核酸适体对靶标的特异性识别和辣根过氧化物酶（HRP）的高效催化反应,发展了一种用于检测三磷酸腺苷（ATP）的酶联核酸适体分析新方法.核酸适体和靶标的特异性结合导致与核酸适体杂交的短链DNA 解链,解离的 DNA 通过杂交被固定在另一酶标板的 DNA 捕获.解离的 DNA 预先标记了异硫氰酸荧光素（FITC）基团, FITC 特异性结合 HRP 标记的 FITC 抗体, HRP 作为信号传导元素催化四甲基二苯胺（TMB）底物显色,通过颜色变化及450 nm 波长处吸光度的变化检测 ATP.该方法对 ATP 具有良好的选择性,检测不受其它物质如 GTP, UTP 和 CTP 的干扰,且检测能在较复杂的试样（体积分数10%和50%的血清）中进行.实验结果表明,在 ATP 浓度为50~400 nmol/ L 范围内,具有良好的线性关系,检出限为26 nmol/ L.
