Background & Motivation to use DENARASE®
The removal of nucleic acids from biological samples is a necessary step for applications in Red Biotechnology and Molecular Biology. Along the production of biopharmaceuticals during fermentative processes – for instance, proteins, antibodies, vaccines, etc. –, nucleic acids are generated and accumulated in the culture broth, particularly when microbial cells are disrupted during the work-up, and intracellular material is poured into the aqueous phase. The accumulation of nucleic acids increases the viscosity of the liquid fraction significantly and complicates the downstream processing and the purification steps. Moreover, to be marketable, and based on strict safety regulations, biopharmaceutical products need to be almost free of nucleic acids.
Conclusively, a nucleic acid removal step is mandatory before commercialization. To tackle that need, traditional approaches comprise sonication, precipitation, or extraction. Though effective, these options often need relatively severe processing conditions (e.g. addition of chemicals or solvents, temperature, pressure, etc.), and thus a partial degradation of the targeted biopharmaceuticals may be expected during the nucleic acid removal step. As an alternative, nucleic acids may be removed upon hydrolysis by means of unselective nucleases, which are hydrolytic enzymes that cleave the phosphodiester bonds of nucleic acids yielding smaller oligonucleotides of several base pairs.
An excellent enzyme candidate is the endonuclease secreted by Serratia marcescens. This endonuclease displays high catalytic activity with a remarkable broad substrate range, efficiently cleaving all forms of nucleic acids, RNA and DNA, single- and double-stranded, as well as linear or circular sequences. The final products are oligonucleotide fragments of 2-5 base pairs. Moreover, the S. marcescens endonuclease shows high stability, remaining active upon the addition of deleterious agents like ionic, non-ionic, chaotropic agents, or denaturing compounds like urea.
Based on its outstanding performance a genetically-engineered form of Serratia marcescens endonuclease – the so-called DENARASE® – has been launched by c-LEcta GmbH (Leipzig, Germany). DENARASE® keeps the expected excellent broad operational window of pH and temperature of the endonuclease of S. marcescens, and besides that, it offers other selling points. The patented production microorganism is not Escherichia coli, but the Gram-positive Bacillus sp., which is known to be an endotoxin-free strain. The employed fermentation media is free of animal-derived feedstocks and antibiotics, and therefore DENARASE® may be considered a BSE/TSE-free product, with a high viral safety (as no animal derived materials are involved in its production). Furthermore, DENARASE® does not display proteolytic activity, and thus does not degrade peptides. This may be relevant when proteins are the marketable targets of the fermentation. Adding to these facts, DENARASE® can be delivered at high quantities and with an excellent quality price ratio. Finally, it must be noted that DENARASE® manufacturing process is in full compliance with the cGMP requirements. Overall, this may open novel markets for endonucleases and may also trigger their use in already established applications, such as medical and (bio)pharmaceutical manufacturing, cell-therapy, oncology, car-t-cell development, etc.
Product & Specifications of DENARASE®
As stated above, DENARASE® is a genetically engineered endonuclease originating from Serratia marcescens. The enzyme catalyzes the hydrolysis of phosphodiesters of single and double stranded, linear, and circular forms of DNA and RNA into smaller oligonucleotides of 2-5 base pairs. The enzyme consists of two protein subunits (each 27 kDa).
DENARASE® shows an optimum temperature of 37ºC. However, it remains active in a much wider range (0-42 ºC) what enables its use in many practical conditions at different temperatures.
Likewise, DENARASE® shows a broad pH range of activity, reaching its maximum in the range of 8-10, regardless the type of buffer employed (MES, Bis-Tris, Tris, CHES, CAPS). These results are a further demonstration of the robustness of the enzyme, able to adapt to many processing conditions.
For its performance, DENARASE® requires Mg2+ as cofactor (1-2 mM). Moreover, the enzyme remains fully active at much higher concentrations of MgCl2 (up to 100 mM). At higher concentrations (> 150 mM), a deleterious effect is observed.
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