| Summary: Acidophilic bacteria from Espiye, Murgul and Zigana has been isolated. Systematical analyses of twelve different isolates were carried out based on 16S rDNA gene sequences. According to 16S rDNA gene sequences, five of the isolates belong to the genus Acidithiobacillus, three of them belong to the genus Acidiphilium and four of them belong to the genus Acidocella.
iro gene coding the iron oxidase of Acidithiobacillus ferrooxidans M1 strain isolated from Murgul cupper mine has been cloned and complete nucleotide sequence was revealed. The gene has been cloned into expression vector pET28(a)+ and overexpressed in E. coli BL21 (DE3). Highly conserved amino acids are determined in iron oxidase and Q9F and S21Y/V22D mutations were performed with site directed mutagenesis technigues at cloned iron oxidase. Wild type and mutant enzymes are purified with HisLink Protein Purification Resin and characterized. Biochemical properties and kinetic parameters of the wild type and mutant enzymes were determined and compared. The wild type optimal temperature was 25°C and maximal activity was observed in pH 4.0. Its Km and Vmax value was calculated as 0,27 ± 0,09 mM and 0,083 ± 0,01 μmol/min/mg protein respectively. The kinetic parameters of the mutant enzymes stayed unchanged. Nonetheless, the maximal activity of the mutant enzymes were moved to pH 3.5 and the pH stability of the S21Y/V22D have increased.
|
