I have to engineer an enzyme which is thermostable but works at pH 7 and above. I want to make it stable at pH 4-6 using in silico analysis. So is there any method to do the same ?
Very briefly,
You should first Know the regions in your enzyme structure which are responsible for its sensitivity to low pHs. If the enzyme structure has an available 3D structure in PDB, that would be possible to compare the amino acid's ionization states in low pH with optimum pH by running Molecular Dynamic Simulations. When you get the idea of which amino acids are probably responsible for the alterations in enzyme function in different pH, you might be able to do mutations in those specific points to get the desired activity. For doing Molecular Dynamic Simulations the following paper might be helpful:
"Short-time dynamics of pH-dependent conformation and substrate binding in the active site of beta-glucosidases: A computational study."
doi: 10.1016/j.jsb.2015.07.002
Hi Vishakha, is your enzyme active at pH 4-6?
Why do you need low pH to do this analysis?
Hi Steingrimur Stefansson, I think its active at acidic pH but it shows optimal activity at alkaline pH in most sources of enzymes. I want to do low pH analysis because when we use this enzyme at industrial scale, low pH is preferred for the successful conversion.
Hi Vishakha, measure its activity versus pH. If it still shows acceptable activity in pH 4-6 range, then I see no reason why not to use it.
The thing I worry about when linking proteins straight onto a solid surfaces is steric hindrance-that is the enzyme active site is inaccessible due to it being so close to the solid surface.
To avoid this, you need to have a spacer arm to link the protein and substrate. For silica substrates, you might want to look into silane spacers.
okay Steingrimur, I will try measuring its activity versus pH but I guess it does show some activity at low pH as well, not optimal though. Regarding spacers, I will have look into it, as I have not used it so far in my work.
Here is a paper describing protein (in this case antibody) immobilization on glass using silanes.
https://www.researchgate.net/profile/Lisa_Shriver-Lake/publication/13778529_Antibody_immobilization_using_heterobifunctional_crosslinkers/links/54de19b70cf2814662ed2332.pdf
Article Antibody immobilization using heterobifunctional crosslinkers
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