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
目的:研究急性应激山羊心律失常发生以及心肌β肾上腺素能受体(-βAR)密度、腺苷酸环化酶(AC)活性和环磷酸腺苷(cAMP)水平的变化,探讨应激状态下交感神经兴奋的细胞信号传入过程。方法:雄性山羊15只,随机分为对照组、应激组和倍他乐克...目的:研究急性应激山羊心律失常发生以及心肌β肾上腺素能受体(-βAR)密度、腺苷酸环化酶(AC)活性和环磷酸腺苷(cAMP)水平的变化,探讨应激状态下交感神经兴奋的细胞信号传入过程。方法:雄性山羊15只,随机分为对照组、应激组和倍他乐克干预组(各5只)。采用声、光复合刺激建立急性应激模型。24 h Holter记录心律失常,留取心房、心室全层肌组织,应用放射性配基受体结合实验、放射免疫分析等方法检测心肌β-AR、AC活性和cAMP水平。结果:①应激诱发情况应激组和干预组山羊出现明显应激表现(燥动不安、敌意,心律失常增多);②β-AR密度与对照组心房、心室肌β-AR密度相比,应激组和干预组显著增高(P<0.05,P<0.01),应激组和干预组间无显著性差异;3组平衡解离常数(KD值)无显著差别;③AC活性应激组心房、心室肌AC水平高于对照组和干预组(P<0.05,P<0.01),干预组高于对照组(P<0.05);④cAMP含量与对照组心房、心室肌cAMP含量相比,应激组和干预组显著增高(P<0.05,P<0.01),干预组cAMP水平低于应激组(P<0.05)。结论:在本研究建立的应激模型中,完整的β-AR-AC-cAMP途径参与了急性应激时交感神经兴奋引发的生物效应。倍他乐克通过对上述信号传入途径的抑制,部分阻断交感兴奋所致的不良反应。展开更多
作者参考 Clement-Cormier 方法,建立了一种简便的组织匀浆腺苷酸环化酶测定方法。该法用非同位素标记的 ATP 代替同位素标记的 ATP 为酶的作用底物,直接用蛋白竞争结合法测定酶的反应产物,cAMP 计算出酶的活性。该法除简化了操作程序外...作者参考 Clement-Cormier 方法,建立了一种简便的组织匀浆腺苷酸环化酶测定方法。该法用非同位素标记的 ATP 代替同位素标记的 ATP 为酶的作用底物,直接用蛋白竞争结合法测定酶的反应产物,cAMP 计算出酶的活性。该法除简化了操作程序外,还可避开同位素标记的 ATP 的不稳定性和较繁琐的分离过程对测定的影响。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.
文摘目的:研究急性应激山羊心律失常发生以及心肌β肾上腺素能受体(-βAR)密度、腺苷酸环化酶(AC)活性和环磷酸腺苷(cAMP)水平的变化,探讨应激状态下交感神经兴奋的细胞信号传入过程。方法:雄性山羊15只,随机分为对照组、应激组和倍他乐克干预组(各5只)。采用声、光复合刺激建立急性应激模型。24 h Holter记录心律失常,留取心房、心室全层肌组织,应用放射性配基受体结合实验、放射免疫分析等方法检测心肌β-AR、AC活性和cAMP水平。结果:①应激诱发情况应激组和干预组山羊出现明显应激表现(燥动不安、敌意,心律失常增多);②β-AR密度与对照组心房、心室肌β-AR密度相比,应激组和干预组显著增高(P<0.05,P<0.01),应激组和干预组间无显著性差异;3组平衡解离常数(KD值)无显著差别;③AC活性应激组心房、心室肌AC水平高于对照组和干预组(P<0.05,P<0.01),干预组高于对照组(P<0.05);④cAMP含量与对照组心房、心室肌cAMP含量相比,应激组和干预组显著增高(P<0.05,P<0.01),干预组cAMP水平低于应激组(P<0.05)。结论:在本研究建立的应激模型中,完整的β-AR-AC-cAMP途径参与了急性应激时交感神经兴奋引发的生物效应。倍他乐克通过对上述信号传入途径的抑制,部分阻断交感兴奋所致的不良反应。
文摘作者参考 Clement-Cormier 方法,建立了一种简便的组织匀浆腺苷酸环化酶测定方法。该法用非同位素标记的 ATP 代替同位素标记的 ATP 为酶的作用底物,直接用蛋白竞争结合法测定酶的反应产物,cAMP 计算出酶的活性。该法除简化了操作程序外,还可避开同位素标记的 ATP 的不稳定性和较繁琐的分离过程对测定的影响。
