Title of Thesis
Biodegradation of Carbamates by soil Bacteria and
Characterization of the methyl Carbamates degradation hydrolase
"med" heterologously expressed in Escherichia coli"
Department of Microbiology / Quaid-i-Azam
|Number of Pages|
|Keywords (Extracted from title, table of contents and
abstract of thesis)|
Biodegradation, Carbamates, Soil,
Bacteria, Characterization, methyl, hydrolase, heterologously,
The present study was aimed to isolate bacteria from soil having capability to degrade N-methylated carbamates in indigenous soil. as well as to characterize the MCD obtained through heterologous expression. Bacteria were isolated from soil having previous history of carbamate application. Twelve different bacterial strains were isolated and screened on mineral salt medium with different concentrations of carbamates (aldicarb, carbaryl and carbofuran and showed good growth up to 1500 ppm concentration. All strains showed capabilities to utilize these compounds as carbon and nitrogen source. These strains were identified as Pseudomonas aeruginosa, Micrococcus arborescens, Brachybacterium sp. Salsuginibacillus kocurii. Brachybacterium and Salsuginibacillus were first time reported to be involved in degradation of N-methylated carbamates.
Degradation of carbamates pesticide in soil was tested. Effect of biostimulation and bioaugmentation were tested in microcosm study where small pots containing soil and pesticide were inoculated with consortium of isolates and soil was amended with nutrients. It was observed that nutrient addition and bacterial consortium had positive effect on the rate of degradation of carbaryl and aldicarb. In microcosms study for carbaryl degradation it was found that in surface soil more than 80% of the parent compound was degraded in treated soil except soil amended with both nitrogen (KNO3) and phosphorus (K2HPO4) sources where only 53% degradation was observed. Same pattern of degradation was observed in case of subsoil (6 inch depth) but degradation was enhanced up to 90%. Results of microcosm study of aldicarb degradation revealed that organic manure (cow dung) proved to be a good stimulant for enhancing the degradation of aldicarb in the presence of selected bacterial consortium. Degradation of aldicarb by bacterial consortium was also observed in liquid medium with initial concentration of 50 ppm. It was observed that optimum pll and temperature for aldicarb degradation was 7.5 and 37oC in liquid culture.
One of the aim of the present study was to express methyl carbamate degrading (mcd) gene of Achromobacter sp. WM111 heterologously in E. coli. In this study we successfully cloned and expressed mcd' in E. coli and purified the enzyme carbamate hydrolase. Methylcarbamate degrading (mcd) gene was amplified, cloned in plasmid pET14b vector and transformed in E. coli BL21 (DE3) cells. The expression of "mcd" in the form of carbamatc hydrolase activity was induced by isopropyl-ß-D-galactopyranoside (IP'I'G). Expression was observed only, at 20oC when tested at different temperature. carbamate hydrolase activity in the transformed cell extract was detected when carbaryl was used as substrate but no activity was detected when aldicarb and carbofuran were used as substrate.
Carbamate hydrolase was purified from the Escherichia coli containing 'mcd gene by passing cell free extract through DEAF and phenyl column using fast pressure liquid chromatography. SDS-PAGE analysis showed that the carbamate hydrolase contained two subunits with molecular weight of 73 and 75 kDa. Characterization of partially purified carbamate hydrolase was performed. Enzyme followed the Michaelis-Menton equation and optimum pllfor enzyme activity was 6.8.
It was observed that after dialysis carbamate hydrolase become completely inactive and its activity was recovered after addition of Mn
or Co++ where Mn++ was better cofactor than Co-- , while addition of copper (Cu'), ferrous (Fe"), ferric ffc+) and zinc (Zn") had no effect on enzyme activity. Optimum temperature for carbamate hydrolase enzyme was 50oC while at 60oC it gave no activity. When stability of the enzyme tested by incubating at different temperatures for 10 min. it was observed that enzyme was quite stable up to 45oC showing 83 % residual activity, even at 55oC more than 50% residual activity was detected while at temperature of 70oC only 30% residual activity was obtained.
MCD obtained through heterologous expression shows significant resistance against the tested organic solvents (methanol, acetone and acclonivile) and is stable up to 25% of all solvents.
Reduetant dithiothreitol (DTT) and oxidant (potassium ferrocyanide) affected the enzyme activity and it was observed that 0.001 mM of DTT increased the hydrolase activity (22%) while oxidant always have negative effect on MCD activity.