How do you search for new enzymes that are more stable for handling, immobilization?

Question

Noob here. I get that I should deduce what characteristics the ideal new enzyme should meet, and then use tools such as PDB and blast to compare to the old enzymes, and use other tools such as pymol, but I have no idea on how to approach this. Where should I look for tutorials?

Answer 1

From your title, you are after better immobilisation, i.e. your enzyme behaves poorly when bound.

• Poor thermostability: Engineering enzymes for better thermostability, involves predicting mutations that improve its Gibbs free energy (∆G, negative potential = better). Finding mutations that improve a protein by going through the mutational landscape is called scanning. Rosetta pmut_scan is a command line tool to do this, while Pross is a really nice webapp that also does it.
• Poor catalysis: if your attachment is close to the active site, that is a design flaw and you need to attach on the other terminus or within a loop. Circular permutation may be an option.
• Aggregation: your enzyme aggregates due to pI close to 7 (see protparam) or has a face that is hydrophobic (see the APBS plugin shipped within PyMOL). Rosetta supercharge is great at suggesting mutations to decrease the pI, but if doing it crudely by changing Gln -> Gln & Asn -> Asp, do check for surface exposure.