Dear Mr. Wizard,

I have been wondering about the effects of falling temperatures during mashing. If I have a single infusion mash that starts high at 155 ºF (68 ºC) (alpha amylase range), and slowly cools back down to 148 ºF (64 ºC) (back into beta amylase range), do the beta enzymes “reactivate” or do the high temperatures cause them to remain denatured?

Steve Antoch
North Bend, Washington

The Wizard Replies:

Denature is one of those words that is often thrown around with little explanation. An enzyme, by definition, is a protein that catalyzes a biochemical reaction. The amylase enzymes catalyze the degradation of starch into smaller molecules.

Enzyme activity is affected by temperature and the rate of a specific reaction increases until the temperature causes the enzyme to denature. In simple terms, the protein or proteins that comprise an active enzyme irreversibly uncoil when they denature. Common examples of denatured protein are found in the refrigerator and kitchen. The cheese making process begins by denaturing milk proteins to form curd. Once milk is converted to curds and whey there is no turning back. Another example of denatured protein is a cooked egg. The solid white material in any cooked egg is denatured egg white. Whether the denatured egg protein is found in meringue, fried, poached or hard-boiled eggs, the native (not denatured) egg white cannot be restored.

Enzymes may be in their native states without being active and environmental factors such as pH, mineral co-factors and substrate concentration do affect enzyme activity. The amylase enzymes active in a mash are most effective around pH 5.2. If the mash pH were increased to say 7 pH by adding a strong base-like sodium hydroxide the activity of the amylase enzymes would dramatically reduce and become inactive. If the pH was then reduced to 5.2 pH by adding a strong acid, the enzymes would again become active. This is because pH affects the electrical charge on the various ionizable groups associated with proteins. These groups include carboxylic acid and amino “functionalities,” to use the scientific language. The charge on a functional group is affected by pH and this has a profound affect on enzyme activity, but pH changes do not necessarily cause an enzyme to denature (although radical changes in pH can cause an enzyme to denature).

The practical message is to be careful with your mash. When in doubt it is always safest to start off a bit cooler than desired and to move the temperature up to the target. Enzyme denaturation does not occur instantaneously and if you intend on mashing in at 140 ºF (60 ºC), for example, and accidentally come in at 162 ºF (72 ºC) you can quickly add cool water and retain the activity of some beta-amylase molecules that have yet to be “cooked” by the high temperature. Happy mashing!