Production and characterization of free and immobilized nitrate reductase from pseudomonas SP. +CD

number: 
1895
إنجليزية
Degree: 
Imprint: 
Biotechnology
Author: 
Shaymaa Hussein Hammoodi Al-Rajehi
Supervisor: 
Dr. Majed H. Al-Gelawi
Dr.Ghazi M. Aziz
year: 
2008
Abstract:

Sixty five soil samples and fifteen water samples were collected from different places in which previously explosions were occurred. Standard techniques yielded ninety six isolates from soil samples capable of growing on minimal media containing 0.1mM TNT or 0.2mM GTN, while the twenty four isolates from water samples un able to grow on minimal media containing 0.1mM TNT or 0.2mM GTN as a sole carbon and nitrogen source. Thirty–nine of the 96 isolates of soil samples were capable to reduce the nitrate to nitrite under aerobic conditions according to the formation of a reddish pink color on tubes. These isolates were classified and characterized as Pseudomonas sp. (19 isolates), Bacillus sp. (12 isolates), Staphylococcus sp. (2 isolates), Acinetobacter sp. (3 isolates), and Micrococcus sp. (3 isolates). Seven isolates, showing the highest reduction of nitrate in minimal media these isolates were selected, classified and coded as SH6, SH7, SH10, SH14, SH17, SH22 and SH31. One Pseudomonas sp. SH7, which was the highest nitrate reduction. To identify if this isolate carry any plasmid that might have a role in nitrate reduction. Plasmid DNA electrophoresis showed that there was no plasmid carried by Pseudomonas sp. SH7, which might lead to thought that the nitrate reduction genes carried on chromosomal DNA. The highest nitrate reductase activity extracted from Pseudomonas sp. SH7 after sonication was 0.98 U/ml compared with Sodium Dodecyl Sulfate method 0.85 U/ml and freezing and thawing method 0.2U/ml. Optimum conditions for nitrate reductase production from Pseudomonas sp. SH7 isolate grown in minimal media containing 0.25mM GTN where investigated and it was found that the initial pH was 7.0 at 35oC for 3 days under aerobic condition, the activity of crude extract was1.5U/ml. Nitrate reductase was purified from Pseudomonas sp. SH7 by several steps including saturation of crude extract with 40-60% ammonium sulphate, ion exchange chromatography by DEAE-cellulose column and gel filtration chromatography through Sephadex G-200 column that revealed overall specific activity of 11.79 U/mg protein, 17.86 fold of purification and yield of 14.9%. SDS-PAGE was used to determine nitrate reductase molecular weight which was about 115 kD. The characterization of partially purified enzyme activity was higher at a pH range between 6.5-7.5 and it showed stability at the same pH range. The maximum enzyme activity was at 35oC and it was stable at 30-40oC for 15 min. While for heat sensitivity it was found that the maximal function 100% activity was observed when incubated for 20 min. at 45oC. Treatment of nitrate reductase from Pseudo. sp. SH7 isolate with 50, 100, 150 and 200 μM sodium azide inhibited the activity by 11, 24, 46 and 76% respectively, whereas 100, 200, 300, 400 and 500 μM potassium cyanide treatment inhibited the activity by 35, 58, 75, 86 and 91% respectively. The enzyme was immobilized by different immobilization methods via entrapment in sodium alginate, agar and sol-gel. It was found that, the sol-gel retained the highest activity (3.5U/ml) while sodium alginate retained (2.8U/ml), but agar method gave about (1.2U/ml). It was found that, nitrate reductase from Pseudomonas sp. SH7, immobilized by sol-gel method had the ability to reduce nitrate to nitrite in soil sample contaminated with 0.5 mM GTN and the enzyme gave a high reduction rate after one hour of reaction ,with a broad pH range from 6.5-10, wider temperature range, and the result showed that the enzyme lost 50% of its activity when it was exposed to 80oC for 15 min., but it retained its activity when it was stored at 4°C after three month storage period.