Understanding The Optimum Temperature For Enzymes

Do you know why Enzymes have Optimum Temperatures? I mean, do you know the chemistry behind it?

It’s a surprising, but simple concept. Read on to discover why it is important to know and how this knowledge could help improve your lab work.

What is the Optimal Temperature for Enzymes?

We use enzymes in the lab for a whole range of reasons. These can span from restriction enzymes for cloning to DNA polymerases for amplifying DNA.

Enzymes from E. coli or warm-blooded animals tend to have an optimum temperature of around 37°C, whereas those from thermal vent bacteria have much higher optimal temperatures. This makes sense when you consider the common temperatures experienced by each. Warm-blooded animals have core temperatures of about 37°C, while bacteria living in thermal vents experience regular temperatures that far exceed this.

However, you might not understand why enzymes have the best catalytic activity within these optimum temperature ranges, but as a biologist it’s good to know the chemistry so here goes…

The Chemistry Behind Optimum Temperatures for Enzymes

Initially, Reaction rate increases with temperature

Chemists have a rule of thumb that a 10°C increase in temperature gives a doubling of the reaction rate. This rule is loosely derived from the Arrhenius equation; as the temperature increases, so does the kinetic energy of the reactants.

This increased kinetic energy means that the reactants are more likely to collide with enough energy to allow the reaction to occur, so the higher the temperature, the higher the reaction rate. So initially – as shown in the green section of the chart in figure 1 – the enzyme activity and hence the reaction rate increases with temperature.

Then it plateaus…

If the enzyme was completely stable even at high temperatures, the reaction rate would continue to increase with temperature until something else happened, like one of the reactants evaporated.

BUT most enzymes are not stable at high temperatures. The have a specific temperature at which thermal denaturation begins to occur. Thermal denaturation reduces the enzyme’s catalytic activity and therefore reduces the reaction rate, and this is the reason for the plateau in the yellow section of figure 1.

Then drops like a stone…

At even higher temperatures (the orange shaded section in Figure 1), the enzyme is more denatured, and lower, or no catalytic activity remains so the reaction rate plummets.

The temperature at which the denaturation occurs depends on the structure of the enzyme, which in turn is related to its evolutionary origin. Thus, E. coli enzymes have evolved to cope with temperatures of around 37°C since they live in the gut. In contrast, enzymes from thermal vent bacteria have been forced to evolve so that they can remain stable at far higher temperatures.

Therefore, an enzyme’s optimal temperature is a trade-off between the Arrhenius-type dependence on temperature (the hotter the reaction, the faster the rate) and the instability of the enzyme as it approaches then reaches its denaturation temperature.

So Why Should You Care About The Optimum Temperature For Enzymes?

Why, as a lab-based biologist, should you worry about the optimum temperature for enzymes? The fundamental reason is that I find biologists (including me) often lack an understanding of the chemistry behind what’s going on in their experiments, which is a serious hindrance, so this article is primarily intended to plug that gap.

But caring about the optimum temperature of the enzymes you use in the lab could also be the difference between experimental success and experimental failure.

Table 1: Optimum working temperatures for commonly used enzymes in the lab.

Optimum temperatures for a range of enzymes commonly used in the lab are shown in Table 1. As you can see, there’s quite a big range, from between 75-80ºC for Taq polymerase down to 25ºC for DNA ligase.

DNA ligase is a great example of why understanding this concept is a good thing for biologists. Typically, we run DNA ligations at room temperature or on ice. Why do we do that if the DNA ligase optimum temperature is 25ºC?

The reason is another trade-off: the efficiency of a ligation reaction relies on the activity of the ligase enzyme, which we know increases with temperature up to 25°C.

But it also depends on the sticky or blunt ends of the DNA being ligated being in proximity to each other so the ligase can do its work. The higher the temperature, the faster the DNA fragments move around in solution so the more difficult it is for the ligase to catch them.

So room temperature is a tradeoff between the ligase activity and slowing down the thermal kinetics ofd the DNA fragments.

But why, I hear you ask, should I perform my ligations at 4ºC if the optimum temperature is far higher? For that, you need to fully understand the process of DNA ligation. Read our article on how DNA ligation works to be reassured.

Originally published July 2016. Reviewed and updated June 2026

Dr Nick Oswald

Nick has a PhD from the University Dundee and is the Founder and Director of Bitesize Bio, Science Squared Ltd and The Life Science Marketing Society.

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