Like all enzymes, restriction endonucleases (REs) need to be handled very carefully to ensure that they do not denature. Which of the following is NOT recommended to protect these fragile structures from denaturation? a. REs should be stored in glycerol solutions, which do not freeze at temperatures below the freezing point of water.

b. Investigators should minimize the number of air bubbles introduced into RE solutions during pipetting.

c. REs should be stored at low temperatures, since the rate of denaturation increases with temperature.

d. REs activity is best preserved by freezing the RE and thawing it out for brief periods of time as needed.

Restriction enzymes can be defined as endonucleases that can recognize specific nucleotide sequence in a DNA (Deoxyribonucleic acid) sequence and cause breakage of phosphodiester bond in the DNA at the target site. Hence, it has the capacity of cleaving a DNA sequence.
These are made up of proteins which are required to be present in the accurately folded conformation in order to function.
Any form of alteration in the three-dimensional structure of the protein induced due to physical, chemical, mechanical or physiological factors can cause denaturation of the protein which in turn can render the protein non-functional.
Freezing of restriction enzymes (REs) directly without the use of glycerol will cause the movement of water molecules within the proteins as well layering of water molecules on the protein surface.
At low temperature these water molecules change to ice crystals which can alter the conformation of the protein or can cause breakage of peptide bonds in the proteins by physical force.
This can cause the protein to get denatured and hence non-functional.
Hence, it is never advisable to preserve REs by freezing directly without glycerol.
Glycerol forms a layer around the protein molecules and prevents the protein molecules from coming into direct contact with the water molecules and hence prevents protein denaturation.