Okeke Benedict C, Frankenberger William T
Department of Environmental Sciences, University of California, Riverside, CA 92521, USA.
Microbiol Res. 2003;158(4):337-44. doi: 10.1078/0944-5013-00213.
Perchlorate (ClO4-) is a major ground water pollutant of public health concern. ClO4- reductase is the key enzyme in the pathway of ClO4- breakdown. ClO4- reductase from cell-free extracts of the ClO4- -respiring bacterium perc lace was purified 10-fold by ion-exchange and molecular exclusion fast protein liquid chromatography (FPLC). The ClO4- reductase catalyzed the reduction of ClO4- at a Vmax and Km of 4.8 U mg protein(-1) and 34.5 microM, respectively. ClO4- reduction was achieved in the temperature range of 20 to 40 degrees C and with optimum activity at 25 degrees C to 30 degrees C and pH 7.5 to 8.0. Molecular masses of two subunits of ClO4- reductase were determined by SDS-PAGE to be 35 kDa and 75 kDa. MALDI-TOF/MS analysis of a trypsin digest of the 35 kDa subunit, revealed several tryptic peptides. Amino acid sequences of 22 tryptic peptides of the 35 kDa ClO4- reductase subunit were obtained by electrospray mass spectrometry. GenBank protein Blast analysis of the amino acid sequences revealed relevant similarity to reductases, dehydrogenases and heme proteins. Data obtained are useful towards the identification of the overall genetic determinants of ClO4- reduction and specific in situ detection of ClO4- as well as NO3-reducing bacteria in ground water.
高氯酸盐（ClO4-）是一种引起公众健康关注的主要地下水污染物。高氯酸盐还原酶是高氯酸盐分解途径中的关键酶。通过离子交换和分子排阻快速蛋白质液相色谱（FPLC），将呼吸高氯酸盐的细菌perc lace无细胞提取物中的高氯酸盐还原酶纯化了10倍。该高氯酸盐还原酶催化高氯酸盐的还原反应，其Vmax和Km分别为4.8 U mg蛋白质-1和34.5 microM。高氯酸盐还原反应在20至40摄氏度的温度范围内进行，在25至30摄氏度以及pH 7.5至8.0时具有最佳活性。通过SDS-PAGE测定高氯酸盐还原酶两个亚基的分子量分别为35 kDa和75 kDa。对35 kDa亚基的胰蛋白酶消化产物进行MALDI-TOF/MS分析，揭示了几种胰蛋白酶肽段。通过电喷雾质谱法获得了35 kDa高氯酸盐还原酶亚基22个胰蛋白酶肽段的氨基酸序列。对这些氨基酸序列进行GenBank蛋白质Blast分析，发现与还原酶、脱氢酶和血红素蛋白具有相关相似性。所获得的数据有助于鉴定高氯酸盐还原的整体遗传决定因素，以及对地下水中高氯酸盐和硝酸盐还原细菌进行特异性原位检测。
