Preparation of ^<13>C-labeled compounds by using photosynthetic microbes and development of its sensitive detection system.

放射性同位素在科学研究中有着广泛的应用，但在安全使用和放射性废物处理方面存在着严重的问题。本课题的目的是利用~（13 C<13>）-CO_2体系培养光合生物，制备~（13 C）-标记化合物<13>，为用~（13 C）-标记化合物替代~（13 C）-标记化合物的研究建立灵敏、选择性高的分析方法<13><14>。以纤细裸藻（Euglena gracilis）为光合微生物，对<13>碳-二氧化碳培养条件进行了初步研究。在光照和CO_2浓度的控制下，对异养和自养生长进行了监测。本研究采用自制的CO_2控制器实现CO_2循环：在自养条件下，裸藻首先<12>在开放系统中通过CO_2-CO_2通道生长。在对数生长期的早期，将^<12>C-CO_2改为^<13>C-CO_2，继续培养至稳定期。在发酵罐内的<13>CO_2浓度控制中，由于没有CO_2监测仪，采用封闭式气路系统<13>对发酵罐内的CO_2浓度进行控制，使发酵罐内的CO_2浓度难以控制。最后，采用开路CO_2，输出的<13>~（13）C-CO_2在碱阱中回收一次，贮存备用，~（13）<13>C标记效率用NMR检测，通过测定葡萄糖在副淀粉中的同位素掺入量来监测。生长后，收获细胞，匀浆并通过离心分级，收集重颗粒副淀粉。目前<13>标记的效率和选择性不高，似乎需要作一些修正，如延长~ <13>1C-CO_2的暴露时间或培养过程中暴露的时间。

Radioisotopes have been widely used in scientific researches, however, there always exist serious problems in the safety usage and the treatment of radioactive wastes. In this project we aimed to produce ^<13>C-labeled compounds by cultivating photosynthetic organisms in the presence of ^<13>C-CO_2, and to establish the alternative way of sensitive and selective analytical methods for the researches using ^<13>C-compounds instead of ^<14>C-labeled ones. Euglena gracilis was chosen as a photosynthetic microbe and the culture conditions using ^<13>C-CO_2 was preliminarily examined. Heterotrophic and autotrophic growth were both monitored, under the control of light and CO_2 concentrations. CO_2 circulation was achieved by a CO_2 controller purchased in this research : in the autotrophic condition, Euglena was first grown by the passage of ^<12>C-CO_2 in the open system. In the early logarithmic phase of growth ^<12>C-CO_2 was changed to ^<13>C-CO_2 and the culture was continued until it reached the stationary phase. During the passage of ^<13>C-CO_2, a closed system of gas circuit was deviced to recover and re-use of waste ^<13>C-CO_2, however, it found difficult to control the concentration of CO_2 without a CO_2 monitor in the jar fermentor. Finally the open circuit of CO_2 was adopted and the output of ^<13>C-CO_2 was once recovered in a alkaline trap and stored until use.Efficiency of ^<13>C-labeling was monitored by NMR by measuring the incorporated isotope into glucose in the cellar particles, paramylon. After growth the cells were harvested, homogenized and fractionated by centrifugation and the heavy particles paramylon were collected. After acid hydrolysis of the particles, the digest was neutralized and applied to the ^<13>C-NMR.At present efficiency of incorporation and selectivity of labeling were not so good that some revision seemed to be required, such as longer exposure of ^<13>C-CO_2 or timing of the exposure during cultivation.