Hi
I want to understand who to extract an enzyme such as a restriction endoneuclease from a bacteria such as E-coli, what is the simplest technique?
The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. For isolation technique go through this article.
Hi Suzanne
If its a recombinant enzyme, you have to make sure that a you included a Tag in your construct.
First, you must lyse the cells either chemically (eg. BugBuster) or physically (eg. sonication).
Second, centrifuge your cell lysate and run supernatant and/or pellet (depends if your enzyme is soluble or in inclusion bodies) on SDS page to verify the presence of the enzyme.
Third, run a chromatography with the specific affinity to elute your enzymes.
Best of luck
Maybe the following papers will help you:
1. Burns B, Mendz G, Hazell S. Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts. Biological Procedures Online. 1998;1:17-26. doi:10.1251/bpo5.
2. Heinis C, Melkko S, Demartis S, Neri D.Two general methods for the isolation of enzyme activities by colony filter screening. Chem Biol. 2002 Mar;9(3):383-90.
3. Jason Chen Kok Ho, Lim Yin Sze; Isolation, identification and characterization of enzyme-producing lactic acid bacteria from traditional fermented foods, Bioscience Horizons: The International Journal of Student Research, Volume 11, 1 July 2018, hzy004, https://doi.org/10.1093/biohorizons/hzy004
First see the enzyme activity in the bacteria, where it is present in the cytosol or in the culture filtrate. Standardize the enzyme assay, follow the standard protocol: ammonium sulfate fractionation followed by passing the active precipitate through phosphocellulose column. Once again determine the enzyme activity and specific activity and then follow it up with hydroxylapatite chromatography. Greene et al Nucleic acid Research. 1978; 5 (7):2373-2380.
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