化学合成独立栄養細菌 Acidithiobacillus thiooxidans 由来アコニターゼの遺伝子解析と大腸菌での発現

An aconitase from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The cloned aconitase gene (acn) was expressed in Escherichia coli JM 109; aconitase activity was found in the cell extarct. The acn gene encodes a 646-amino acid polypeptide and is located upstream of the isocitrate dehydrogenase gene (icd). A. thiooxidans aconitase showes high sequence similar to pig heart aconitase and E.coli aconitase B. Twenty-five of twenty-seven active site residues assigned in pig heart aconitase are conserved in A. thiooxidans aconitase. The enzyme was purified by DEAE-Toyopearl 650M column chromatogrophy. The purified enzyme had an optimum pH of 7.5 and an optimum temperature of 60 C. Thermal inactivation studies of the purified enzyme revealed the enzyme activity to be uninfluenced after one hour incubation at 40 c. Enzyme activity was retained 100% after incubation of the enzyme at pH 6.0-9.0 for 60min. The A. thiooxidans aconitase was composed of a single polypeptide chain with a molecular mass of 66 kDa.