Pseudomonas sp. MN5의 특성과 망간산화단백질 정제

Abstract

충청남도 병천면 일대의 6곳의 토양시료를 채취하여 망간을 산화하는 균주들을 순수분리 하고, 이 중 망간 산화능이 가장 우수한 한 균주를 최종 선별하여 본 실험에 사용하였다. 최종 선별된 균주의 생리, 생화학적 특성을 조사하고, 16S rRNA 염기 서열분석 등을 통하여 동정한 결과 최종 선별된 균주는 Pseudomonas sp. MN5로 확인되었다. Pseudomonas sp. MN5은 fructose와 maltose를 제외한 다양한 당을 이용하지 못하였으며, 항생제인 kanamycin, chloramphenicol, streptomycin 그리고 tetracycline에는 높은 감수성을 보이고, 리튬, 망간, 바륨과 같은 중금속에 대해서는 mg/ml 단위의 높은 내성을 나타냈다. 그리고 Pseudomonas sp. MN5의 망간산화 최적 pH는 7.5이고, 망간산화 활성이 proteinase K와 가열처리를 한 시료에서 저해되었다. Pseudomonas sp. MN5가 생성하는 망간산화 단백질을 ammonium sulfate precipitation, HiTrap Q FF ion exchange chromatography 그리고 G3000sw <TEX>$_{XL}$</TEX> gel filtration chromatography를 통해서 정제한 결과, 15 kDa, 46.7 kDa 그리고 63.5 kDa의 세종류의 manganese oxidizing protein가 확인되었고, 내부서 열과 N-말단 서열 분석 결과 Pseudomonas sp. MN5가 생성하는 망간산화 단백질은 외막의 porin 단백질인 것으로 추정되었다. Bacterial colonies which were able to oxidize the manganese were isolated from six soil samples in Byungchon area. Among them, one bacterial strain was selected for this study based on its high manganese oxidation activity. This selected bacterial strain was identified as Pseudomonas sp. MN5 through physiological-biochemical test and analysis of its 16s rRNA sequence. This selected bacterial strain was able to utilize fructose and maltose, but they doesn't utilizing various carbohydrates as a sole carbon source. Pseudomonas sp. MN5 showed a very sensitive to antibiotics such as kanamycin, chloramphenicol, streptomycin and tetracycline, but a high resistance up to mg/ml unit to heavy metals such as lithium, manganese and barium. Optimal manganese oxidation condition of Pseudomonas sp. MN5 was pH 7.5 and manganese oxidation activity was inhibited by proteinase K and boiling treatment. The manganese oxidizing protein produced by Pseudomonas sp. MN5 was purified by ammonium sulfate precipitation, HiTrap Q FF anion exchange chromatography and G3000sw <TEX>$_{XL}$</TEX> gel filtration chromatography. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, three manganese oxidizing protein with estimated molecular weights of 15 kDa, 46.7 kDa and 63.5 kDa were detected. Also, it was estimated that manganese oxidizing protein produced by Pseudomonas sp. MN5 were a kind of porin proteins through internal sequence and N-terminal sequence analysis.