DENARASE® High Salt

Mfr.No.	22002
Description	Product Characteristics Like the wild-type S. marcescens endonuclease, DENARASE High Salt is capable of the nonspecific cleavage of phosphodiester bonds between the nucleotide subunits of nucleic acids. The enzyme can hydrolyze all forms of DNA and RNA, such as single-stranded, double-stranded, linear, circular, or supercoiled. The protein consists of two monomers with a calculated molecular weight of 27 kDa each. The calculated isoelectric point (pI) of DENARASE High Salt is at pH 7.83. Magnesium (Mg2+) is an essential co-factor and prerequisite for the nucleic acid clearance activity of the salt-tolerant endonuclease. Production Process DENARASE High Salt is a technical enzyme produced using the established DENARASE manufacturing process based on recombinant expression in a Bacillus sp. production host. The gram-positive production strain is free of endotoxins. DENARASE High Salt is manufactured and supplied by c-LEcta GmbH, the owner of the patented production process. Compliance DENARASE High Salt is intended for use in research and development (R&D) applications and process development of biologicals such as viral vectors and vaccines. R&D-grade DENARASE High Salt is produced under ISO 9001 standard, without the use of antibiotics, Triton X-100 and raw materials of animal origin or TSE/BSE risk. A DENARASE High Salt GMP-grade for clinical applications will become available as soon as possible. Please contact your local supplier for more information on the projected launch date of the GMP Grade. The manufacturing process of the DENARASE High Salt GMP-grade will comply with the same regulations and standards as our well-known wild-type GMP-grade DENARASE products. This includes manufacturing under EU GMP conditions acc. to EU GMP regulations and distribution compliant with EXCiPACT and ANSI/NSF 363 Standard. Thus, the requirements for Good Manufacturing and Distribution Practices (GMP/GDP) for pharmaceutical excipients are also met. From a technical performance perspective, DENARASE High Salt R&D- and GMP-grade will be equal.
Purity	The protein purity is determined by SDS-page and silver staining. The purity of the protein must be at least 99 %.
Formulation	DENARASE High Salt is supplied in a stabilizing glycerol solution with the following specifications: 50 % Glycerol v/v 20 mM Tris-HCl, pH 7.4 ± 0.2 250 mM NaCl 5 mM MgCl2
Notes	To minimize the risk of denaturation during storage and transport the enzyme is formulated in a stabilizing glycerol solution
Activity	Like other enzymes, the activity of DENARASE High Salt depends on various conditions including temperature, pH and concentration of inhibiting compounds. Furthermore, the DNA clearance activity of endonucleases is strongly influenced by the nature and concentration of the substrate. To properly compare the activity of two DENARASE High Salt lots or salt-tolerant endonucleases from different suppliers, it is important to strictly follow the same protocol. For DENARASE High Salt activity analysis, we use the following assay: Substrate buffer: 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA, 1 mg/mL DNA (see also Table 1 for a reference list of chemicals used). Note: For the preparation of substrate buffer, 50 mg DNA is added to 32 mL 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM MgCl2, vortexed for 5 s and incubated at 80 °C for at least 12 hours to dissolve the DNA. After cooling to room temperature, the DNA concentration is determined photometrically. The final substrate buffer is prepared by diluting in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM MgCl2 to a final concentration of 1 mg/mL DNA and addition of BSA to a final concentration of 0.1 mg/mL. DENARASE High Salt is diluted prior to use in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA to obtain values within the linear range of the assay. Typically, values within the linear range are obtained for samples with 200-300 U/μL with a dilution of 1:30,000 and for samples with 300-400 U/μL with a dilution of 1: 40,000. After equilibration of substrate buffer at 37 °C, the reaction is started by adding 125 μL diluted DENARASE High Salt or “buffer blank” (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM MgCl2, 0.1 mg/mL BSA) to 2.5 mL substrate buffer followed by incubation at 37 °C. After exactly 15, 30, 45 and 60 min, 500 μl samples are drawn and immediately inactivated by adding 500 μL 4 % perchloric acid. Acidified mixtures are incubated on ice for at least 45 minutes and centrifuged subsequently (10 minutes at ~ 17,000 x g). The amount of acid soluble nucleotides in the supernatant is measured at 260 nm. The activity of the enzyme is specified in Units. One Unit is defined as the amount of enzyme that causes a change in absorbance at 260 nm of 1.0 Absorbance Unit in 30 minutes (see also the formula below for precise calculation of activity). Note: Vendor specifications for the activities of various commercially available salt-tolerant endonucleases cannot be compared due to differences in the activity assays that are used.
Video

Applications Range	DENARASE High Salt enables optimal and cost-effective removal of nucleic acids in biomanufacturing processes that benefit from higher salt concentrations. The enzyme is especially suitable for the use at bioprocessing-relevant 200-500 mM salt and pH 7.4 – pH 8. It is designed to improve the efficiency of various applications: Viral Vaccines Viral Vectors for Cell & Gene Therapies Viscosity reduction in Lysates Sample preparation in Electrophoresis and Chromatography.

Reference	/

Storage	DENARASE High Salt is stable at a storage temperature of -20 °C ± 5 °C within specification range for a period of at least 6 months from the date of QC release. Accordingly, long-term storage is recommended at -20 °C ± 5 °C. Note: It is not recommended to store the product at -70 °C or below, since freezing the product will cause loss of activity. The long-term stability of DENARASE High Salt is currently under investigation.
Shipping	DENARASE High Salt is filled in non-pyrogenic, USP Class VI compliant vials. The product vials are shipped under qualified cooled conditions. Shipping temperature may differ from storage temperature without affecting product quality. All DENARASE products will be delivered by c-LEcta in a sealed secondary packaging with tamper-evident seals.
Shipping Condition	DENARASE High Salt is shipped by default in insulated boxes with cold packs as a precautionary measure to protect against unexpectedly elevated temperatures during transport. Storage at -20 °C ± 5 °C is recommended upon receipt of the product. To address concerns regarding DENARASE High Salt stability at elevated temperatures during transport, delays in shipping or custom clearances and/or refrigeration problems at the customer locations, the following stability tests were conducted: Incubation of DENARASE High Salt I) at 4 °C for 3 months II) at room temperature for 28 days III) at 30 °C for 3 days No loss of activity was observed for tests I – III after indicated time periods. This confirms that short-time storage at 4 °C, room temperature and 30 °C will not negatively impact DENARASE High Salt activity. Consequently, no adverse effect on DENARASE High Salt activity is to be expected in the event of a short-term failure in cooling during transport. The data further demonstrate that DENARASE High Salt can be stored over several days at temperatures deviating from recommended storage conditions without impacting product quality.

Protocals And Manuals	DENARASE High Salt Validation Guide CoA 25 kU CoA 100 kU CoA 500 kU CoA 1000 kU CoA 5000 kU

DENARASE High Salt Group Shot

Fig. 1 The effect of increasing NaCl concentrations on the enzyme activity of the wild-type S. marcescens endonuclease (DENARASE) and DENARASE High Salt. The volumetric activities at 15 mM MgCl2 and pH 7.4 as well as pH 8 were measured in U/ml. After normalization to the activities as given on the CoAs of the respective enzyme batches, enzyme activities were normalized to DENARASE High Salt activity at 250 mM NaCl and pH 7.4 (indicated by dotted circle). The yellow and pink hatched areas mark the recommended salt range for the use of DENARASE and DENARASE High Salt, respectively.

Fig. 2 The effect of increasing NaCl concentrations on the enzyme activity of DENARASE High Salt and Salt Active Nuclease High Quality (SAN, ArcticZymes). The volumetric activities at pH 7.4 (left) and pH 8 (right) were measured in U/ml. After normalization to the activities as given on the CoAs of the respective enzyme batches, enzyme activities were normalized to DENARASE High Salt activity at 250 mM NaCl, 15 mM MgCl2 and pH 7.4 (indicated by dotted circle).

Fig. 3 Effect of low to high MgCl2 concentrations on enzyme activity of DENARASE High Salt at 250 mM NaCl. The volumetric activities at pH 7.4 and pH 8 were measured in U/ml and normalized to activity at 5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

Fig. 4 Effect of low to intermediate MgCl2 levels in combination with 0-500 mM NaCl on enzyme activity of DENARASE High Salt. The volumetric activities at pH 7.4 (left) and pH 8 (right) were measured in U/ml and normalized to activity at 250 mM NaCl, 5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

Fig. 5 Effect of MgCl2 on DENARASE High Salt activity in Tris-HCl and potassium phosphate (KP) buffers. Volumetric activities at 250 mM NaCl were measured in U/ml and normalized to maximal enzyme activities. DENARASE High Salt activity has been measured in frequently applied buffer systems such as Tris-HCl and phosphate buffers (Fig. 5). For all tested buffers the DENARASE High Salt activity was enhanced by increasing MgCl2 concentrations, which thus can be a solution to circumvent the inhibitory effects of high phosphate concentrations.

Fig. 6 Effect of temperature on DENARASE High Salt activity. Enzyme activity at different temperatures at pH 7.4 and 8.0 were normalized to activity at 37 °C, 5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

Fig. 7 Effect of pH on DENARASE High Salt activity. Enzyme activities in Tris-HCl, Bis-Tris and potassium phosphate (KP) buffer at different pH levels in the presence of 5 mM and 15 mM MgCl2 were normalized to DENARASE High Salt activity at 5 mM MgCl2 and pH 7.4 (indicated by dotted circle).

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Available in US inventory. Typically dispatched within 2-3 business days.

Combination SKU	Supplier No.	Size	Your price
BHZ16500002-25K	22002-25k	25 kU (25,000 Units)	$310	Add to Cart
BHZ16500002-100K	22002-100k	100 kU (100,000 Units)	$780	Add to Cart
BHZ16500002-500K	22002-500k	500 kU (100,000 Units)	$2380	Add to Cart
BHZ16500002-1000K	22002-1000k	1 MU (1,000,000 Units)	$3470	Add to Cart
BHZ16500002-5000K	22002-5000k	5 MU (5,000,000 Units)	$11740	Add to Cart

DENARASE + DENARASE High Salt FAQ

What can DENARASE^® be used for?
DENARASE^® specifically hydrolyses the phosphodiester bonds between nucleotides, leaving smaller fragments of approximately 3-5 base pairs. The enzyme is active on all forms of nucleic acids, including single-stranded, double-stranded, linear, circular or supercoiled. DENARASE^®-mediated removal of unwanted residual plasmid and host cell-derived nucleic acids can significantly improve the purification and yield of viral vectors and proteins. It is therefore widely used in research, process development and production of biologicals such as viral vectors and vaccines.

How should we add DENARASE^®to our process?
There are several publications on how to use endonucleases derived from Serratia marcescens, such as DENARASE^®. We recommend starting with concentrations between 10 and 60 U/mL. Several parameters such as time, concentration and temperature are highly dependent on your process conditions and should be evaluated accordingly.

Is DENARASE^®active at higher salt concentrations?
The optimum salt concentration for DENARASE^®is < 20 mM for monovalent ions. As shown in Figure 7 of the DENARASE^® Product Information Sheet (DENARASE PIS), enzyme activity decreases with increasing salt concentration (KCl & NaCl). We therefore suggest that the concentration of monovalent cations such as Na⁺ and K⁺ should be kept below 200 mM for optimal DENARASE^® activity.

Is there any risk for endotoxins?
Endotoxins are lipopolysaccharide-protein (LPS) complexes that are part of the outer membrane and are released during the lysis of Gram-negative bacteria such as E. coli. They are normally detected via the Limulus Amebocyte Lysate (LAL) test. The production host of DENARASE^® is a Gram-positive Bacillus strain, classified as GRAS (Generally Recognized As Safe) and not releasing LPS. The risk of endotoxins originating from the production strain is therefore very low. The general risk of endotoxin contamination, e.g. coming from process raw materials, devices, environment, etc., is carefully assessed, resulting in consistently low endotoxin levels of < 0.25 EU/kU on the specification (as evaluated by LAL testing; see page 4 DENARASE PIS).

How can DENARASE^® be removed from my process?
A variety of methods are available to remove DENARASE^® from target products. Depending on the characteristics of the process, anion exchange, cation exchange, hydrophobic interaction, hydroxyapatite or size exclusion can be applied. Alternatively, filtration techniques can be used for specific process solutions. Depending on the target molecule, the type of chromatography or filtration media must be tested for the specific case.

Are there different quality grades for DENARASE^®?
DENARASE is available in two quality grades: DENARASE^® for research and development (R&D) use and DENARASE^® for manufacturing under GMP. GMP-grade DENARASE^® is manufactured under EU GMP conditions and provided with dedicated regulatory support for market approval e.g. via an own US FDA Drug Master File. R&D-grade DENARASE^® is produced in conformity with ISO 9001 standard with less strict requirements regarding documentation, storage and distribution. The R&D-grade is suitable for R&D stages, when fast and easy access to raw materials is key. From a technical performance perspective, both quality grades are equal and the specification parameters are the same.

How can we detect residual DENARASE^® in our process solution?
DENARASE^® can be detected using commercially available Serratia marcescens endonuclease ELISA kits. For the quantitative analysis of residual S. marcescens endonucleases - including Benzonase® Nuclease (registered trademark of Merck KGaA) - in your process samples, we offer our DENARASE^®ELISA Kit. Based on highly specific monoclonal antibodies, our kit provides unmatched reproducibility and the lowest endonuclease detection and quantification limits compared to similar products.

DENARASE® High Salt

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