Difference between revisions of "Part:BBa K3519000"

To examine antibiotic degradation, the culture was set in 50ml minimal media at 30 °C and cell-free cultures were used as control. To monitor adsorption of sulfonamides heat-killed cells were used. The culture was centrifuged at 13000g for 20min and the supernatant obtained was separated and analyzed using HPLC.

Crude extract assay

Sulfonamide-grown cells were disrupted by sonication in 50 mM Tris-HCl buffer (pH 7.5). Cell debris was removed by centrifugation at 13,000 ×g at 4 °C for 1 h and filtered (0.2 μm) to obtain cell-free protein extracts. The reaction mixture contained 250 μg of protein, 200 μM sulfonamide, 1 mM NADH, and 5 μM FMN in 1 mL of 50 mM Tris-HCl buffer (pH 7.5) and incubated at 30 °C. The reaction was stopped by adding 12% phosphoric acid. Samples taken from the reaction mixture were analyzed by HPLC. To detect metabolites in the reaction, the reaction mixture was extracted with an equal volume of ethyl acetate three times. The ethyl acetate extract was evaporated to dryness under nitrogen gas. The residue was dissolved in methanol for HPLC analysis.

Activity assay for recombinant strains

E. coli BL21(DE3) harboring appropriate plasmid constructs were grown at 37 °C overnight in 5 mL of LB medium supplemented with 100 mg/L of ampicillin. The overnight culture was transferred into 50 mL of fresh LB medium containing ampicillin and incubated at 30 °C. Isopropyl-β-D- thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM when the cells reached an OD600 of 0.4–0.6 and the culture was induced for 3 h. Sulfamethoxazole was added to the culture at a final concentration of 0.5 mM and the culture was further incubated for 16 h. The culture supernatant was taken at intervals for HPLC analysis.
In the presence of NADH and flavin cofactor (FMN or FAD), the heterologously expressed and purified sulfonamide monooxygenase and flavin reductase of strain CJ77 (Fig. 1) resulted in the rapid degradation of sulfonamides with concomitant production of the dead-end products and 4-aminophenol in stoichiometric manners.In the present study, the mechanism of the initial cleavage reaction of sulfonamide drugs was demonstrated using these purified enzymes. (Fig. 2)


Fig. 2. Proposed sulfonamide degradation mechanism for the initial cleavage reaction mediated by sulX (Sulphonamide monooxygenase) and SulR (flavin reductase).[3]

Cloning, expression, and purification of monooxygenase and flavin reductases

Cloning and expression of genes were conducted using pET28-(a) vector for single gene expression and pETDuet-1 vector for co-expression of two genes in E. coli BL21 (DE3). Using appropriate primers Monooxygenase and flavin reductase genes were PCR amplified.. E. coli BL21(DE3) harboring appropriate plasmid constructs were cultivated at 37 °C. At an OD600 of 0.5, cultures were induced with IPTG at a final concentration of 0.1 mM, and then further incubated at 37 °C for 3 h or at 20 °C for 12 h as required. Cells were harvested and re-suspended in 20 mM Tris-HCl buffer (pH 7.5). Cell-free protein extracts were obtained as described previously. The recombinant His- tagged proteins were purified using His GraviTrap column.

Enzyme assay for kinetic studies

Sulfonamide degradation activity by purified enzymes was determined by HPLC analysis as described above. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM, flavin reductase, 1.0 μM FMN and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. The reaction was stopped by adding 12% phosphoric acid and sulfamethazine and its metabolites were quantified at every 1 min for 5 min. ( Fig 2). Steady state kinetic parameters were obtained by fitting initial velocity data to the standard Michaelis-Menten equation. The initial velocities for various concentrations of sulfonamides were obtained with sulfonamide monooxygenase (0.5, 5.0 and 2.5 μM), the equivalent amounts of flavin reductase and FMN, and 200 μM NADH at 25 °C for 1 min.(Fig 3).

In the presence of NADH and flavin cofactor (FMN or FAD), the heterologously expressed and purified sulfonamide monooxygenase and flavin reductase of strain CJ77 resulted in the rapid degradation of sulfonamides with concomitant production of the dead-end products and 4-aminophenol in stoichiometric manners.



Fig. 3. UV–visible spectrum during the sulfamethazine degradation by purified proteins. The reaction mixture contained 50 μM sulfamethazine, 2 μM sulfonamide monooxygenase, 0.1 μM flavin reductase,2.0 μM FMN, and 200 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. Scan was taken at every 30 s for 5 min[3].



Fig. 4. Kinetics of the sulfonamide degradation reaction. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM flavin reductase, 1.0 μM FMN, and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and incubated at 25 °C. Sulfamethazine (closed circle), 2‑amino‑4,6‑dimethylpyrimidine (open square) and 4‑aminophenol (open triangle) were analyzed over time[3].

1.3. Antibiotic susceptibility test:

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