Purification and Characterization of Nitrate Reductase (NAR) from Pseudomonas sp. SH7 Isolate

Abstract

Sixty five soil samples and fifteen water samples were collected from different places in which previously explosions were occurred in Iraq. Seven isolates showed ability to utilize 0.1mM trinitrotoluene (TNT) and/or 0.2mM glycerol trinitrate (GTN) as a sole carbon and nitrogen source and one of these isolates showed the highest nitrate reduction which was classified and coded as Pseudomonas sp. SH7. The highest nitrate reductase activity extracted by sonication while optimum conditions for enzyme production in minimal media pH 7 containing 0.25mM GTN at 35oC for 3 days under aerobic condition. Nitrate reductase was purified by 40-60% ammonium sulphate, ion exchange and gel filtration. Nitrate reductase molecular weight determined by SDS-PAGE was 115 kD. The characterization of purified enzyme activity and stability was higher at a pH between 6.5-7.5 and. Maximum activity was at 35oC and stable at 30-40oC for 15 min., while for heat sensitivity 100% activity observed at 45oC for 20 min. Treatment with 200 µM azide and 500 µM cyanide inhibited the activity by 76 and 91% respectively.