以高热稳定性的腺苷酸基琥珀酸合成酶为催化剂大量合成腺苷酸基琥珀酸(盐)  

Large-scale synthesis of adenylosuccinate using a high thermal stability adenylosuccinate synthetase as a bio-catalyst

作　　者：王楠 姜允嘉 王洋 赵晓宏 郭鹏 蔡大勇 谢勇 WANG Nan;JIANG Yun-jia;WANG Yang;ZHAO Xiao-hong;GUO Peng;CAI Da-yong;XIE Yong(Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100193, China)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所中草药物质基础与资源利用教育部重点实验室,北京100193

出　　处：《中国医药生物技术》2019年第2期108-114,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(81473114)

摘　　要：目的实现腺苷酸基琥珀酸(盐)(S-AMP)的大规模合成,为开展药物学等研究提供原料。方法以pET-28-a为表达载体,利用大肠杆菌表达古细菌Pyrococcus horikoshii OT3来源的腺苷酸基琥珀酸合成酶(PhAdSS),利用Ni-NTA层析柱纯化后作为催化剂在实验室内开展这种微生物体内合成S-AMP的反应。用Bradford法测定纯化后PhAdSS的浓度。利用硅胶薄层层析检测反应进度。用硅胶柱层析法和重结晶法纯化S-AMP,利用质谱法测定合成品中S-AMP的分子量,利用紫外分光光度法测定合成品内S-AMP的含量及回收率。结果经纯化后从1 L的自动诱导培养基中至少获得20 mg的His-tagged-PhAdSS。将含有10 mmol/L肌苷酸、11 mmol/L L-天冬氨酸、20 mmol/L鸟苷三磷酸、4 mmol/L MgCl_2、2.9μmol/L的His-tagged-PhAdSS溶液于常压环境中,70℃恒温6 h以上可以实现IMP完全转化为S-AMP。纯化后可得到S-AMP的单晶体,利用紫外分光光度法测定的纯度为94%,收率为17%。结论实现了PhAdSS为催化剂的S-AMP的大量合成。Objective To set up a large-scale synthesis system of adenylosuccinate (S-AMP) for providing mass chemical resources in pharmaceutical studies. Methods Adenylosuccinate synthetase (AdSS) from archaea Pyrococcus horikoshii OT3 (PhAdSS) was expressed in E.coli cells using the pET-28a vector as a His-tagged fusion protein. The His-tagged-PhAdSS was purified by a Ni-NTA column. Concentration of the His-tagged-PhAdSS was determined using the Bradford method. The His-tagged-PhAdSS was used as a catalyst to perform large-scale synthesis of S-AMP in vitro. Analysis of the reaction progress was carried out using the silica gel thin-layer chromatography. S-AMP was purified using the silica gel column chromatography and re-crystallization. The molecular weight of synthesized S-AMP was measured using mass spectrometry. The concentration of S-AMP in the synthesized sample and yield of the large-scale synthesis system were determined using the UV spectrophotometry. Results More than 20 mg His-tagged-PhAdSS could be obtained from per liter of auto-induce medium. Under a normal pressure environment, IMP can completely be converted to S-AMP in a solution containing 10 mmol/L inosine mono-phosphate (IMP), 11 mmol/L L-aspartate, 20 mmol/L guanosine tri-phosphate (GTP), 4 mmol/L MgCl2 and 2.9 μmol/L His-tagged-PhAdSS, with gently stirring at 70 ℃ for more than 6 hours. After purification, single crystal of S-AMP was obtained. Yield and recovery rate of the large-scale synthesis system of S-AMP set up in this study were 94% and 17%, respectively. Conclusion A large-scale synthesis system of S-AMP is set up using PhAdSS as a bio-catalyst.

关 键 词：腺苷酸基琥珀酸(盐) 腺苷酸基琥珀酸合酶 古细菌Pyrococcus horikoshii OT3 

分 类 号：R915[医药卫生—微生物与生化药学]