Hi,

it would be helpful to see the actual data.

I actually had similar problem in the past. The problem was, that my crude extract used up my cofactor (DTT), so it wasn't available for the reaction. Similarly, you may have some inhibitor of your activity - the fungus doesn't want to be digested by its own enzyme, right? You may try to partially purify your enzyme (through one or two chromatography columns) and check, whether it behaves better. If so, you can simply set up conditions such that you do not observe much of the inhibition and go for it consistently.

Thanks for your response. We can review the data you've obtained so far. I'm having trouble getting a reliable progress curve. I used the "salting-out" technique to purify my enzyme of interest and separate other proteins. I used different amounts of my sample, and even using a control enzyme like trypsin, the response doesn't change. What could be going on?

If the crude extract contains multiple proteolytic enzymes with different substrate characteristics, the rate of reaction could depend on the extent of the degradation of casein at any given time. Different enzymes in the crude extract could prefer different sites on the casein molecule. Also, as the casein molecule undergoes proteolysis, additional new sites are exposed that are favored by some enzymes over others. Thus, the combination of a crude extract containing multiple proteolytic enzymes with a substrate that becomes increasingly heterogeneous as the reaction progresses leads to a nonlinear reaction rate over time and a nonlinear relationship between the enzyme concentration and the reaction rate. I think the linearity would be better if measured at short times and with a high substrate concentration, although in that case you might be observing the activity of only a subset of the enzymes present.

To the biochemist Arthur Kornberg is attributed the aphorism "Don't waste clean thoughts on dirty enzymes."