| Summary: | 碩士 === 國立彰化師範大學 === 生物技術研究所 === 97 === Aphid is an important agricultural pest insect feeding on the phloem sap of plants. As phloem sap lacks nitrogenous compounds, the growth of aphid relies on the endosymbiont Buchnera aphidicola for providing essential amino acids. Thus, the Buchnera enzymes for biosynthesis of the essential amino acids are ideal and safe targets for pesticides. N-acetylglutamate synthase (NAGS, encoded by argA) is a good target that plays an important role in the arginine biosynthesis pathway. Buchnera can not be cultivated in vitro. The relationship between Buchnera and E. coli is close since both belong to the Enterobacteriacae family. Therefore, expression of Buchnera NAGS in an E. coli system is a good avenue to study the function of Buchnera NAGS and its regulatory mechanisms. In this study, we constructed a plasmid that contains Buchnera argA, and transformed the plasmid into the E. coli argA mutant NK5992 for complementation test. Our results demonstrated that the Buchnera argA complemented for arginine auxotroph at 20℃, 28 ℃, but not at 36 ℃. Buchnera NAGS was detected in transformed E. coli BL21(DE3) cells by SDS-PAGE. The enzyme was precipitated with 20 ~ 40 % saturation of ammonium sulfate, and purified to homogeneity by metal-chelating affinity and ion-exchange chromatography. Its activity was measured using a DTNB-based method. In our result, Buchnera NAGS splited acetyl group from acetyl-CoA without glutamate in vitro. The Km value for the substrate acetyl-CoA was 449.73 μM in the present of 10 mM glutamate. The Km value for the substrate acetyl-CoA was 544.85 μM without glutamate. The activity of this enzyme was not inhibited by arginine, with only 6.56 % of its activity inhibited in the present of 5 mM arginine.
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