I want to purify keratinase enzyme from LB broth and I am looking for the best method. Can any one suggest one?
You should concentrate the broth by ammonium sulfate or ultrafiltraration, and then you can use an affinity column or an ion exchange column
Ammonium sulphate precipitation is the best method. Use different concentrations of ammonium sulphate ranging from 30-70% to get optimum quantity followed by dialysis.
Doing the following procedure, concentrate your crude preparation by ammonium sulfate or ultrafiltraration, then based on the property of this enzyme, you can choose ion exchange chromatography, such as DEAE-cellulose; otherwise, you can use hydrophobic chromatography, such as Phenol-agarose, subsequently, concentrate your collected fraction by ultrafiltraration, and purify it by gel filtration chromatography(Sephacryl S-200).
Good luck!
After removing biomass precipitate protein by ammonium sulphate (30-70%). remove salts by dialysis.
Depending on the pI of the protein, you may also do an ion exchange chromatography as a first step. For this, you will need to use LB with the lowest possible NaCl concentration or dilute the culture medium after the biomass has been removed. When diluting, you should include buffer to bring adjust the pH to a value that is 2 units away from the pI of your protein. Depending on the charge of your protein, you next add DEAE sepharose or CM Sepharose or similar matrices and stir the beads with the protein-containing solution (magnetic stirrer in coldroom). Let the beads settle and remove the supernatant. When most of the supernatant is gone, fill the beads in a column and elute the protein with increasing NaCl concentrations. Works especially fine when your protein has an extreme pI.
First choose effective production medium (with keratin as substrate) for keratinase production, Usually you wont get much on LB.
Do keratinase activity assay with the culture filtrate.
ammonium sulphate precipitation method will be used for initial extraction of keratinase.
Finally column purification (Sephadex) based on your protein molecular weight you can choose.
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