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Benzonase® Endonuclease FAQs

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Working with Benzonase® endonuclease for the first time – or using it in a new application? Here are answers to the most frequently asked questions about Benzonase® endonuclease.

Which quality/quantity of Benzonase® endonuclease will be adequate for a certain application?
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.

At which step do I have to introduce Benzonase® endonuclease in my process?
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.

How much more Benzonase® endonuclease do I have to add if I am working at low temperatures?
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.

Why is Benzonase® endonuclease not working? What will inhibitits activity?
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.

I observe a loss of activity - why?
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.

My Benzonase® endonuclease was left out on the bench all weekend. Is it still good?
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.

How do I inhibit Benzonase® endonuclease activity?
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.

How do I remove Benzonase® endonuclease?
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.

Is Benzonase® endonuclease safe?
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.

Is Benzonase® endonuclease free of protease activity?
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.

Is Benzonase® endonuclease compatible with protease inhibitor cocktails?
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.

In which step should I introduce Benzonase® endonuclease to my process?
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.

Do you offer immobilized Benzonase® endonuclease?
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.

Why is the filling range volume of the 5-million-unit tubes not specified?
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).

If you don’t find what you’re looking for here, please view our detailed brochure or get in touch with your local MilliporeSigma representative.

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