I am doing High Throughput Screening HTS of functional metagenomic library to screen for cellulose-degrading enzymes using fluorogenic substrate, I want to know what is the best lysis solution for recombinant E.coli and what do we need to add in the buffer? (Some research are using Potassium Acetate pH 5.5).
Cordially!
https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/
This might be helpful. For enzyme isolation, to preserve the activity, I would recommend the physical disruption methods like the sonication or freezing and grinding. Also, this way you can also use the ideal buffer for cellulose-degrading enzymes to resuspend your cells. For example, 50 mM potassium acetate buffer.
In my lab we are using two ways of cell lysis for high-throughput screening in the microtiter plate format: i) permeabilization by detergents (e.g. Triton X-100, BugBuster) and/or enzymes (e.g. lysozyme); ii) freezing and thawing (often one cycle is sufficient according to my experience). Please check the paper attached for more details.
You should choose the buffer system and pH, where most known cellulases are active (unless you would like to find atypical cellulases).
Article Fully automatized high-throughput enzyme library screening u...
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