Why are restriction enzymes kept in 50% glycerol solution?

Although restriction enzymes are typically purchased in slightly larger batches, they are used in VERY small quantities.

If nothing else, you usually want to perform multiple tests on the same batch; as a result, the batch that was purchased must be kept in storage for a considerable amount of time.

The standard limit is 24 hours, but most enzymes are perfectly happy in their buffer at 4 degrees Celsius for a while before degrading.

The batch must be frozen for longer term storage; -20C is the standard and will keep it fresh for several months; -70C is the standard for even longer periods (e.g., one or more years).

It must be frozen quickly and in small containers (like Eppendorfs) to prevent frequent thawing and refreezing.

Stabilisers like DTT (di-thio-treitol), serum albumin, and—most importantly—glycerol (5–50%) can/should be added to prevent damage to the protein at such low temperatures.

Oddly enough, 50% glycerol must be used at -20C, but at -70C, glycerol is not really required, though adding it can help stabilize the protein.

Restriction enzymes are kept in a 50% glycerol solution to prevent their degradation and maintain their stability during storage. Glycerol acts as a cryoprotectant, protecting the enzymes from freezing damage, and helps maintain enzyme activity over time.