Enzymes by post—restriction enzyme stability

To the editor:

DNA restriction enzymes, with their widespread applications in many diagnostic procedures (e.g., RFLP, PCR, etc), are the very cornerstone of recombinant DNA technology. Unfortunately, their transport and storage are usually carried out at subzero temperatures, greatly increasing the cost per unit activity and limiting availability in many developing countries.

While preservation techniques to extend stability at higher temperatures have been reported^1,2,3,4 (e.g., desiccation with trehalose), enzymes are generally unavailable in this format, and storage at −20°C following reconstitution is still recommended. Surprisingly, no one appears to have examined the stability of untreated restriction enzymes as supplied by manufacturers. Our studies suggest that restriction enzymes are in fact quite stable as supplied; some exhibited activity after several weeks' storage at 37°C, and some lost little or no activity after storage for several months at ambient temperatures (J. Clark, J.C. Harrison, and J.B. March, unpublished data). We have even sent enzymes roundtrip between the UK and USA with a marginal loss in activity (Figure 1), a surprising finding given the large fluctuations in temperature likely to occur during such a journey (September 1999).

Figure 1: Ethidium bromide-stained agarose gels of restriction digests of 0.5 μg λ-DNA, using 1 μl of serially diluted restriction enzymes (in supplied 1× buffer).

HindIII, EcoRI (Boehringer Mannheim), SmaI (Promega). All enzymes at 10 U μl⁻¹. In the lower lanes (Airmail), DNA was digested with restriction enzymes that had been sent by conventional airmail in a plain envelope from Edinburgh, UK, to Boston, MA, and returned via the same route. In the upper lanes (−20°C) digestion was performed with samples of the same enzymes stored at −20°C over the time course. The apparent stability of SmaI is particularly surprising given that it is considered thermolabile, with an incubation temperature of 25°C often recommended.

If applicable to all enzymes, these findings could have major implications. Air transport of dry ice packages is very expensive, restricting their availability in Third World markets; at present, the cost of carriage can easily exceed the purchase price of many enzymes. However, we successfully used inexpensive airmail letters to send restriction enzymes to Tanzania. Upon arrival, they retained activity and were effectively stored in a domestic refrigerator for months. At present, the supply of such reagents involves expensive overland transport on ice from South Africa. By reducing carriage costs and removing the need for refrigerants and insulation materials, simple overnight postage should yield considerable savings while benefiting the environment.

Widespread availability of affordable enzymes in the Third World could have a major impact on both research and diagnostic activities. We would therefore urge manufacturers to, first, investigate the stability of their own enzymes at higher temperatures, and, second, consider the direct supply of enzymes to hitherto uneconomic African and Asian markets. Even if a percentage loss of activity occurs during transport, enzymes are usually used in such excess that compensation can easily be achieved either by recalibrating activity upon arrival or extending incubation periods (e.g., overnight). We have examined more than 20 enzymes (J. Clark, J.C. Harrison, and J.B. March, unpublished data), and all exhibit significant activity after one week at room temperature, while some (e.g., HindIII and Tsp509I) still exhibit activity after storage for six months at 37°C. Although we have only tested restriction enzymes, these findings may well be applicable to other classes of enzymes, particularly thermostable polymerases. A more detailed investigation of a greater range of restriction enzymes by the manufacturers themselves would be beneficial to those in the research community, especially those in countries where enzymes are currently difficult or impossible to obtain because of transportation or storage constraints.