Benzonase® Endonuclease FAQs

There are several parameters which influence the activity of Benzonase® endonuclease. Hence, the optimum conditions will vary from process to process and need to be determined experimentally. For viscosity reduction, Benzonase® endonuclease, purity grade II (≥90%) will often be sufficient.

The answer to this question will vary depending on why you are using Benzonase® endonuclease. The example applications given in our brochure will hopefully help you answer this question. However, as a general rule, Benzonase® endonuclease is usually best added after the fermentation step and before the capture step.

At temperatures below 37°C the efficiency of Benzonase® endonuclease decreases. The amount needed to compensate for this decrease in efficiency will vary from process to process and on the other parameters present. Often, increasing another parameter, such as incubation time, can compensate without needing to increase the quantity of Benzonase® endonuclease used.

Benzonase® endonuclease is active under a wide range of operating conditions, however a concentration of 1–2 mM Mg²⁺ is essential for the activity of Benzonase® endonuclease. Mn²⁺ can substitute Mg²⁺, however the enzyme will only reach its optimum activity in the presence of Mg²⁺. It is inhibited (approximately 50% activity) by monovalent cation concentrations >300 mM, phosphate concentrations >100 mM, and by ammonium sulfate concentrations >100 mM. In addition, concentrations of >1 mM EDTA will also inhibit Benzonase® endonuclease activity.

Benzonase® endonuclease is usually very stable, however in rare cases a loss of activity can be observed. There are several possible reasons for this; irreversible inactivation can be due to the presence of denaturing agents in the sample, e.g. proteases, or alternatively due to incorrect storage. Reversible inactivation is commonly due to the presence of chelating agents such as EDTA, which remove essential magnesium ions.

We have done extensive stability testing on Benzonase® endonuclease, and find that it is extremely stable. Even with extended incubations at 37 °C, Benzonase® endonuclease maintained >90% of activity for several weeks. After a storage time of 11 weeks at 25°C (60% RH) the activity even remained unchanged.

There are process additives/agents that affect Benzonase® endonuclease activity – for example it can be inhibited by high salts, like >300 mM monovalent cations, >100 mM phosphate, >100 mM ammonium sulfate, >100 mM guanidine HCl. Other known inhibitors are chelating agents, like EDTA, which could cause loss of free Mg²⁺-ions (EDTA concentrations >1 mM have shown to inhibit the enzymatic reaction). This can be reversed by adding more MgCl₂.

Removal of Benzonase® endonuclease can be accomplished by several downstream units of operation like depth filtration for clarification, tangential flow filtration (TFF) for concentration & diafiltration and chromatography (IEX, SEC, HIC). Please see our brochure (page 22) for further information.

Yes, toxicological studies with Benzonase® endonuclease have been performed (internal reports available). The systemic toxicity after single application was investigated in mice and rats: no toxic effects have been observed even at very high doses. In addition no mutagenic potential has been observed in mice treated intravenously even with a very high dose of Benzonase® endonuclease.

Yes, Benzonase® endonuclease is supplied without detectable protease activity and is hence not degraded during its “work.” The presence of protease in the sample itself will, however, result in irreversible degradation of the Benzonase® endonuclease.

Yes. However, caution should be exercised since many protease inhibitor cocktails include EDTA. Concentrations of greater than 1 mM EDTA will inhibit the activity of Benzonase® endonuclease.

This varies depending on the application. However, as a general rule, Benzonase® endonuclease is usually best added after the fermentation step and before the capture step.

No. All efforts to bind Benzonase® endonuclease to a support that meets the demands of a commercial product with respect to activity, stability, and regulatory requirements have so far been unsuccessful.

As the specific activity (U/ml) of Benzonase® endonuclease may vary between production lots, we decided to specify the units per tube but not the volume. The volume per tube can be easily calculated from the specific activity information on the Certificate of Analysis (CoA).