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

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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. "