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Understanding The Optimum Temperature For Enzymes

Posted in: Basic Lab Skills and Know-how
thermometer to depict optimum temperature for enzymes

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Do you know why there is an optimum temperature for enzymes? Does it even matter? 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 narrow temperature ranges. It’s reassuringly simple, and we will explain more below, where you will also find out why understanding the optimum conditions for your enzymes could mean the difference between experimental success and failure!

Why Do Enzymes Have Optimum Temperatures?

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 and is depicted in Figure 1. 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. 

Understanding The Optimum Temperature For Enzymes
Figure 1: The relationship between reaction rate and temperature to help explain the optimum temperature for enzymes.

The first part of the reaction rate profile (shown shaded in green in Figure 1), where the rate is increasing with the temperature, follows the Arrhenius equation. To put it another way, the higher temperature, the maximum activity of the enzyme! 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.

The reaction rate begins to plateau and then falls in the yellow highlighted section of the graph in Figure 1. This is due to the temperature approaching the point at which the enzyme begins to undergo thermal denaturation (and therefore, the protein structure is damaged, causing the enzyme to lose activity).

At even higher temperatures (the orange shaded section in Figure 1), the enzyme is fully denatured, and no activity remains.

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. 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?

This theoretical chemistry is all well and good, but why, as a lab-based biologist, should you worry about the optimum temperature for enzymes? To put it simply (and rather dramatically!), caring about the optimum temperature of the enzymes you use in the lab could be the difference between experimental success and experimental failure!

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


Optimal Temperature (°C)

Taq polymerase


DNA ligase




Restriction enzymes


Optimum temperatures for a range of enzymes commonly used in the lab are shown in Table 2. As you can see, there’s quite a big range, from between 75-80ºC for Taq polymerase down to 25ºC for DNA ligase.

It’s therefore important to always check the manufacturer’s guide for the optimum temperature for your particular enzyme. If not, and you accidentally use too high a temperature, you risk denaturing the enzymes and losing their desired activity.

So, for example, if you incubate your DNA ligase enzyme with your cloning fragments at a higher temperature than the optimum, no correct ligation productions for you! Alternatively, if you run a PCR with a Taq polymerase at a temperature below the optimum, you won’t get the maximum possible activity out of your enzyme. This could result in low yields of PCR product! It’s important you use the optimum temperature for enzymes in your lab work to ensure you’re getting their best activity and, therefore, the best yields of the desired product!

Why Enzymes Have Optimum Temperatures Summarized

The optimum working temperature of an enzyme is a because of the interplay between chemistry, where higher temperatures equal faster reactions, and biology, where proteins become denatured at certain temperatures.

Enzymes from thermophilic organisms, such as Taq polymerase, have higher optimal temperatures, as they have adapted to survive in temperatures with extreme heat. To work in such hot environments, enzymes of thermophilic organisms need to remain active (and therefore not denature) at these high temperatures.

Understanding the optimal working temperature of the enzymes you are using will allow you to optimize your experiments to yield maximum results.

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 October 2022.

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  1. Thasindu Gunaratne on March 13, 2017 at 8:36 am

    Adding one more thing, as the temperature gets closer to ideal, doesn’t the reaction time get faster? And if so, Why is this?

  2. Tarun on October 20, 2007 at 10:47 am

    Nice to know that. Thank you!

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