Functional expression of RubisCO by a high-speed carbon dioxide fixing microorganism

高速二氧化碳固定微生物对 RubisCO 的功能表达

• 批准号: 07455325
• 负责人: KODAMA Tohru
• 金额: $ 4.86万
• 依托单位: Shinshu University (1996)The University of Tokyo (1995)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07455325/
• 关键词: Rubis CO; CO_2 fixation; イオンクロマトグラフ; specificity factor; 活性化

A thermophilic hydrogen bacterium Pseudomonas hydrogenothermophila strain TH-1 possesses cbbQ gene under RubisCO gene (cbbLS). CbbQ protein was thought to participate in the post-translational activation/stabilization of RubisCO.We showed the presence of cbbO gene under cbbQ gene and clarified the gene sequence. In the next step, plasmids harboring cbbQ,cbbO,cbbQ-cbbO gene were prepared. By using one of the above plasmids and a plasmid harboring cbbLS,transformation of E.coli was performed. Then, difference in activity or expression of RubisCO was analyzed by measuring RubisCO activity or by electrophoreis, respectively. When only the plasmid harboring cbbLS was used, RubisCO activity was low and the RubisCO protein was detected as a smear band on polyacrylamide-gel electrophoresis. Contrary to this, in the presence of cbbQ or cbbO gene, the activity of RubisCO increased 2 to 3 fold, also the RubisCO protein was detected as a clear band on polyacrylamide-gel electrophoresis.In a separate experiments, three kinds of protein (CbbLS,CbbQ,and CbbO) were prepared for the experiment of in vitro RubisCO activation. However, successful results have not been obtained.During our research, CbbO protein itself was proved to possess CO_2 incorporation activity, which will be a source of thinking for future research.

一种嗜热产氢细菌Pseudomonashydrogenothermophila菌株TH-1，在RubisCO基因（cbbLS）下具有cbbQ基因。CbbQ蛋白被认为参与RubisCO的翻译后激活/稳定，我们发现cbbO基因存在于cbbQ基因下，并阐明了基因序列。在下一步中，制备含有cbbQ、cbbO、cbbQ-cbbO基因的质粒。通过使用上述质粒之一和携带cbbLS的质粒，进行大肠杆菌的转化。然后，分别通过测量RubisCO活性或通过琼脂糖凝胶电泳分析RubisCO活性或表达的差异。当仅使用携带cbbLS的质粒时，RubisCO活性较低，并且在聚丙烯酰胺凝胶电泳上检测到RubisCO蛋白为涂片条带。与此相反，在cbbQ或cbbO基因的存在下，RubisCO的活性增加了2 - 3倍，并且RubisCO蛋白在聚丙烯酰胺凝胶电泳上被检测为清晰的条带。在本研究中，我们发现CbbO蛋白本身具有CO_2参入活性，这为进一步的研究提供了思路。

项目成果

Yokoyama et.al.: "Genes encoding RubisCO in P.hydrogenothermophila are followed by a novel cbbQ gene similar to nirQ" Gene. 153. 75-79 (1995)

Yokoyama 等人：“嗜氢假单胞菌中编码 RubisCO 的基因后面跟着一个类似于 nirQ 的新型 cbbQ 基因”基因。

Yaguchi et.al.: "A Non radasotopic Anion-exchange Chromatographic method to omeasure the specifiafy factor" Biosci.Biotech.Biochem.,. 60(印刷中). (1996)

Yaguchi 等人：“测量特定因子的非雷达同位素阴离子交换色谱方法”Biosci.Biotech.Biochem., 60（出版中）。