If you get your protein expressed in good amount, it suggests your protein cannot be easily digested by proteases. Otherwise, most of the expressed protein would be digested during expression.

Your protein won't get digested by column process because when it binds to column, it is separated from other proteins. Unless your wash and elusion buffer contain proteases, which I assume is highly unlikely.

There are many factors can affect kinetic assay. You cannot sure that your bad kinetic curve comes from degraded enzyme unless you have proof that your enzyme is degraded. Have you run SDS gel to check your purification?

Cydi from EZBiolab

Kindly have a look at the image there,

It looks like you have quite good expression if the marked band is your protein. What the gel looks like after the protein is eluted from the column?

Cydi from EZBiolab

That is the eluted protein

So your protein did not get purified.

I am wondering if that is the case. But when I did Enzymatic kinetics with the lysate, it worked, giving the typic Menten curve

this can only tell that your enzyme is present in the reaction mixture but cannot tell its purity

It is a good idea to carefully note the total protein (mg), the specific activity (Katal/mg) and the total activity (Katal) after each purification step. You should also do a PAGE, making sure that each lane corresponds to the same volume of cytosol. So, if you had, say, 50 mL cytosol, and used 10 µL for PAGE, and your column eluate is 2 ml, then use 10 µL * 2 mL / 50 mL = 0.4 µL for PAGE. That way, you can easily compare the recovery and purification factor of each step.

Some protease "inhibitors" like PMSF are actually inactivators, these you need to add only to the cytosol as they permanently kill Ser-proteases. True inhibitors need to be present as long as proteases have not been removed, at whatever step that happens in your case.

Another possible problem is that some proteins become unstable when purified. In that case, carrier protein like BSA can help.

Oh, and I assume that your enzyme is soluble!

Honestly, I think you have too many issues. Protease degradation is probably the least you should concern at this stage. For example, the marked strong band might not be your target protein after all if you did not have a way to verify it. pET expression system always give a band at thus position. The activity you observed might not come from your enzyme given how crude you sample was. You need to 1) confirm the band is the enzyme by western or other method 2) purify the enzyme to reasonable pure and then measure activity.

"don't waste a clear idea by a dirty enzyme", Do you know who said this?

Cydi from EZBiolab