Please ,I work with fermentation of whey isolate with lactobacillus strains ,I would like to measure the cell lysis rate depending on releasing of intracellular peptidase , So I have to measure this on whole cell suspension or on the cell free supernatant of fermented media?
Also what is the normal protein range I should have to calculate the proteolytic enzymes specific activity on whey isolate and MRS ?
Thanks
Dear Eman:
In a similar posting I had given a rapid protocol for a similar enzyme. It is like this: After you have homogenized the cells at 4C centrifuge the suspension at 10000 rpm for 15 minutes to remove all cell debris. Now you may saturate this with 80% ammonium sulfate again at 4C and leave it overnight. Centrifuge the suspension at 8000 rpm for 15 minutes at 4C. This is your crude protein containing the enzyme. Dialyse this against 25mM phosphate buffer again over night (preferably ) at 4C. Now carry out ion exchange chromatography using DEAE cellulose with 0.1M NaCl gradient with 0.5M NaCl. Collect minimum 50 fraction of 10 ml each. All these has to be carried out at 4C. Calculate the protein content at 280nm for each fraction and do the enzyme assay using casein as substrate and plot the graph of protein content and enzyme activity for all 50 fractions. Now run native PAGE and SDS Page with MW markers. Check enzyme activity for probable bands on Native PAGE. Use only those fraction having significant protease activity. This will be sufficient for enzyme characterization. Depending upon your work, you may do trypsin digestion of the band and carry out AA sequence from the C and/or N end of the peptides if you wish to do computational Biological studies.
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