Freezing will at least slow down the reaction, although it will probably resume when you thaw the mix. Changing the pH to a value outside the range in which your enzyme is active would be the most common way, but this would fall under the category "chemicals". You could use your enzyme immobilized on beads and , depending on the kind of beads, spin them down, filter them off or remove them with a magnet to separate the enzyme from the reaction product.

To give more specific advice, we would need to know more details about the specific enzyme/reaction and scale of the process.

Freezing will at least slow down the reaction, although it will probably resume when you thaw the mix. Changing the pH to a value outside the range in which your enzyme is active would be the most common way, but this would fall under the category "chemicals". You could use your enzyme immobilized on beads and , depending on the kind of beads, spin them down, filter them off or remove them with a magnet to separate the enzyme from the reaction product.

To give more specific advice, we would need to know more details about the specific enzyme/reaction and scale of the process.

You may separate the enzyme and products using column chromatography, for example on size or charge. Alternatively you may try toinactivate the enzyme by protease activity.

I agree with Annemarie, immobilised enzyme or freezing.

If your enzyme and the substrate are sufficiently differing in molecular weight, an additional method present itself group separation (commonly known as desalting).

If your substrate is small molecule, then NAP-5, NAP-10 or PD-10 columns can be used.

Alternatively, if 2 proteins rational selection of gel filtration media based on exclusion limit of the matrix can be used to separate 2 proteins rapidly using a self pack column.

I wonder if when you say "stop the enzyme after enzyme modification" you mean stop the modification of the enzyme by some chemical modifier. If so, the only way of stopping the reaction of an enzyme with a chemical modifier is to decrease the concentration of the chemical modifier by a) dilution; b) dyalisis or c) sephadex chromatography (provided the modifier has a low molecular weight). A variety of the latter procedure is the technique of "centrifuge columm (Sephadex)" described by Penefsky (J.Biol.Chem. 252, 2891 - 2899, 1977) for desalting an enzyme and for measuring the binding of Pi. This method permits to eliminate the modifier from the reaction medium only slightly decreasing the enzyme concentration (an advantage over a) a c) procedures) and is much faster (few minutes) than b) and c).

It would help to have more specific info, if for example all chemicals are forbidden or only some. Chemicals used for stopping come from the classes of acids, bases, detergents and specific poisons (e.g., Hg for enzymes with catalytic -SH groups).

If you want to do it completely without chemicals and heat, I could only think of spin columns: fill a tuberculin syringe 2/3 with Sephadex, put a frit or a little sand on top and let any excess fluid drain. Use the wings on top of the syringe to hang it into a short test tube. When you want to stop the reaction, place up to 0.2 mL of the solution on top of the column and immediately spin in a table-top centrifuge for a few seconds. The enzyme will leave the column with the void volume, small molecules will stay in the gel pores. The pore size of the Sephadex has to be selected accordingly.