1- Experiment 4: Ampicillin Production
i. Step for ampicillin production by using 1 pot two steps method (1P2S)
- In order to prepare penicillin G solution 40mM, we dissolve 0.143g of penicillin powder in 10ml PB (pH= 7, 0,1M).
- We take 7.5ml of this solution and we place it in beaker 100ml.
- We put 0,8g iPGA ( or about 99,2 UpenG/gram of carrier).
- Stirr (T˚= 22-25˚c / for 60min).
- Here we have 6-APA with PAA in the solution.
- After 60min we prepare a D-PGME solution (120mM) by dissolving 0,24g of powdered D-PGMEH in 10 ml PB (pH= 7, 0,1M).
- We take 7.5 ml of the preceded solution that will be added in the beaker where the reaction is happened.
- Then we add 0.24g of iPGA (or 30 UpenG/ gram of carrier).
- We adjust pH from 6.4 to 7 (with NaOH, 1M).
- We wait for about 1270min (22.5h) till the reaction will be achieved.
- The total time needed to produce ampicillin is 1350 min
i. Ampicillin purification and harvesting
- After the reaction will be finished, H2SO4 is added in order to stop the enzymatic reaction.
- Then the solution is filtrated to discard the enzyme from the filtrate
- 1ml is took in Eppendorf to testing the effectiveness of our produced ampicillin against E.coli which is a sensitive strain against ampicillin
- Then a solvent is added to the residual solution (ethyl acetate)with ratio 14:2 (each 14ml of filtrate need 2ml of solvent) in order to make ampicillin soluble in ethyl acetate -----> Here we have obtained two phase
- We discard the aqueous phase and we conserve the organic phase in a tube
- Sodium bicarbonate is added with ratio 10:1.44 to the medium ( 1.44 g of sodium bicarbonate is added to 10ml) in order to obtain a solid phase.
- The last step is to filter and dry the residue to obtain ampicillin powder as ampicillin sodium. Her the produced ampicillin is ready to be tested and quantified.
ii. Ampicillin qualification:
- To quantify our produced ampicillin, the obtained filtrate took in a last step is adding to the surface of a standard agar plate
- After 2 or 3 minute we discard the surplus of the filtrate from the surface then a strain of E.coli sensitive to ampicillin is streaked to the surface of medium
- After 24 hours of incubation at 37oC the result is mentioned below