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Permeabilization of gram-negative bacteria with KPi/hexane for the release of L-asparaginase

Source: Hikmet Geckil's Lab
Date Added: Wed Oct 22 2003
Date Modified: Tue Oct 28 2003
Abstract: This is a fast, efficient and reproducible recovery procedure for periplasmic L-asparaginase from distinctly related gram-negative bacteria, Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa. As the method uses inexpensive organic solvent hexane and an aqueous salt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. As hexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation. Enzyme recoveries up to 3-fold with respect to sonication can be achieved with this system.

  1. Harvest (10,000 rpm for 5 min) 24 h grown (in LB pH 7.5, 37 oC, 200 rpm) Escherichia coli, Enterobacter aerogenes or Pseudomonas aeruginosa cells.
  2. Wash once with 0.05 M potassium phosphate (KPi) bufferpH 8.6, and resuspended to A600= 5.0 in the same buffer with 1 % hexane.
  3. Incubate the suspenssion at room temperature for 1 hour, briefly wortexing for every 10 min.
  4. Leave the tube caps open for 5 min in order to evaporate volatile upper phase (hexane) prior to analysis of L-asparaginase activity in the cell-free aqueous phase.
  5. Prepare cell suspensions also in distilled water or 0.05 M KPi with no hexane as controls.
  6. Measure the enzyme activity utilizing the Nesslerization reaction. This method utilizes the determination of ammonia liberated from L-asparagine in the enzyme reaction by the Nessler reaction. L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia.
  7. Start the reaction by adding 0.1 ml permeabilized cell suspension or 0.1 ml supernatant of cell suspensions from aqueous phase into the pre-warmed 0.9 ml 0.01 M L-asparagine prepared in 0.05 M Tris-HCl buffer, pH 8.6 and incubate for 30 min at 37 0C.
  8. Stop the reaction by the addition of 0.1 ml 1.5 M TCA.
  9. Centrifuge the reaction mixture at room temperature (10,000 rpm for 5 min) to remove the precipitate.
  10. Determine the ammonia released in the supernatant colorimetrically (A480) by adding 0.25 ml Nessler reagent into tubes containing 0.5 ml supernatant and 1.75 ml dH2O.
  11. Vortex the content and leave at room temperature for 10 min.
  12. Read the A480 values against the blanks that received TCA before extract addition.

One L-asparaginase unit (U) is defined as the amount of enzyme that liberates 1 mmole of ammonia per min at 37 0C. Specific activity is expressed as units per milligram of protein released. The ammonia concentration produced in the reaction can be determined from the standard curve obtained with ammonium sulfate as the standard. The limit of detection of ammonia by this method is about 10 mM. Determine the total protein colorimetrically by any standard method available, using bovine serum albumin or any other commercially available protein as the standard.
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