I am preparing Enzymatic Antibiotic API by using Penicillin G Acylase Enzyme in aqueous medium but the problem is that after the condensation reaction is complete, the reaction goes backward by hydrolysis of the final product very fast.
It is very difficult to control the reaction. Can anyone suggest how to stop the hydrolysis?
Why do you think that the reaction is complete and then the reaction goes backward rapidly?. Normally, the system will tend to reach equilibrium and it will be never complete.....
To add on to Francisco's answer... The equilibrium can be pH dependent and the reverse or forward reaction can be favored at low or high pH. One can drive the reaction in the direction they desire by adjusting pH.
A couple of things to think about..
1. Stopping the reaction once the condensation rxn is complete. I would recommend using small spin filters that work in a micro centrifuge (bench top) to stop the reaction by removing the enzyme, they come in different molecular weight cut-offs (10, 20 , 30Kda) etc...
2. Maybe the hydrolysis is non-enzymatic- removing the enzyme will help you understand if this is enzymatic or non enzymatic hydrolysis.
Hope some of these thoughts help..
Saturating the solution with the substrate could let you recover more condensation product. Alternatively, it may be worth exploring aqueous two phase systems with Poly(ethylene glycol) aiming at separation of the reaction product from the enzyme.
If it is realy enzymatic reaction YOU can inhibit the enzyme. e.g. by extreme pH's or some chemicals.
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