Improvement of CO2-fixaton in cyanobacteria by transforming NADH oxidase system

我们以前纯化的酶系统（NADH氧化酶-AhpC），其功能作为过氧化物酶和氧化酶从有氧生长的Amphibacillusxylanus，缺乏呼吸链和过氧化氢酶。该酶体系对过氧化氢和烷基过氧化氢均有极高的清除活性。本发明的最终目的是提高叶绿体固定CO_2的能力，其中光合作用中形成的过量氧和过氧化氢抑制固定CO_2的反应。为了建立一种有效的清除叶绿体中过量氧和过氧化氢的方法，进行蓝藻的生化研究和nadh氧化酶ahpc基因在蓝藻中的表达研究，本研究以蓝藻为研究对象，建立了一个叶绿体模型。将AhpC基因克隆到pTrac 99 A中，在大肠杆菌中高效表达。杆菌2.集胞藻AhpC的催化性质利用过氧化物酶活性测定系统，对集胞藻AhpC的催化活性进行了研究。木聚糖NADH氧化酶在集胞藻的无细胞提取液中，尽管NADH氧化酶的活性很低，但仍有很高的活性，这表明集胞藻中的Ahpc是由不同的酶还原的，表达的NADH氧化酶不会与集胞藻中的还原酶系统竞争。coli和集胞藻中表达，并将nadh氧化酶-ahpc基因片段克隆到载体中。将该载体以10 4 /μM DNA的转化效率转化集胞藻，但集胞藻中不表达NADH氧化酶-AhpC蛋白。我们正在构建集胞藻表达系统，在集胞藻中表达NADH氧化酶-AhpC蛋白。"

We previously purified an enzyme system (NADH oxidase-AhpC) which functions as peroxidase and oxidase from aerobically grown Amphibacillus xylanus that lacks both respiratory chain and catalase. The enzyme system showed a extremely high scavenging activity for both hydrogen peroxide and alkyl hydroperoxide. The final purpose of the application is to improve the ability of CO2-fixation in chloroplast, in which exess oxygen and hydrogenperoxide formed in photosynthesis inhibit CO2-fixing reacton. In order to establish an effective elimination method of the excess oxygen and hydrogenperoxide in the chloroplast, biochemical investigations and the expression of nadh oxidase ahpc gene in cyanobacteria, a model of chloroplast is performed in this application.1.Expresstion of AhpC gene of Synechocystis sp. PCC6803 in E. coli The gene of AhpC has been cloned into pTrac99A and overexpressed in E. coli. The recombinant enzyme has been purified to homogeneity.2.Catalytic properties of Synechocystis AhpC.The catalytic activity of Synechocystis AhpC was examined by the peroxidase assay system with A. xylanus NADH oxidase. Regardless of a very low activity coupled with NADH oxidase, distinctly high activity was observed in cell free extract of Synechocystis, suggesting that different enzyme from Axylanus NADH oxidase reduces AhpC in Synechocystis and the expressed the NADH oxidase will not compete with the reduction enzyme system in Synechocystis.3.Transformation and expression of nadh oxidase-ahpc gene in Synechocystis.We have constructed a shuttle vector system between E. coli and Synechocystis and the fragment containing nadh oxidase-ahpc gene was cloned into the vector. Though the vector was transformed into Synechocystis at transformation efficiency 10 4 /μM DNA, NADH oxidase-AhpC protein was not expressed in the Synechocystis. We are reconstructing an expression system of Synechocystis to express NADH oxdase-AhpC protein in the Synechocystis. "