DENARASE® | Upgrading the Endonuclease from Serratia marcesce

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Appropriate handling and removal of nucleic acids is crucial for many applications in red biotechnology and molecular biology. Upon the production of many biopharmaceuticals in fermentative processes (e.g.,proteins, antibodies, and vaccines), nucleic acids are concomitantly generated and accumulated in culture broth, particularly when microbial cells are disrupted during work-up.
Such accumulation increases the viscosity of the liquid fraction significantly and complicates downstream processing and purification. To be marketable and comply with strict safety regulations, biopharmaceutical products need to be nearly free of nucleic acids. Therefore, a nucleic acid removal step is mandatory before commercialization of (fermented) biopharmaceuticals is considered.

How can nucleic acids of a biological sample be removed without degrading a marketable product? Some traditional approaches include sonication, precipitation, and extraction strategies, which have been demonstrated in different applications dealing with fermentative units. However, such options often require relatively severe processing conditions (e.g., addition of chemicals or solvents, temperature, pressure). Thus, a partial degradation of targeted biopharmaceuticals can be expected to occur during the nucleic acid removal step. That can lead to unaffordable downstream units and to unacceptable losses of production efficiency and purity.

As a highly relevant alternative, enzyme technology can provide creative and efficient solutions for nucleic acid removal under milder and more biocompatible conditions than those of other methods. Nucleic acids can be removed with hydrolysis using unselective nuclease enzymes.

In a broad sense, nucleases are hydrolytic enzymes that cleave phosphodiester bonds of nucleic acids, yielding smaller oligonucleotides of several base pairs. In nature, nucleases control genetic quality through their involvement in DNA replication and reparation. Remarkably, nucleases can degrade different nucleic acids present in a biological sample. Within this group of enzymes, unspecific nucleases are promiscuous catalysts that can cleave all types of nucleic acids: single- or double-stranded, linear or circular, DNA or RNA and without predefined sequence specificity. Unselective performance makes such enzymes very useful for nucleic acid removal in practical applications.

In this field, an excellent enzyme candidate is the endonuclease secreted by Serratia marcescens, a gram-negative bacterium. This endonuclease shows high catalytic activity with a remarkable broad substrate range, efficiently cleaving all forms of nucleic acids, RNA, and DNA (single- and doublestranded as well as linear or circular sequences). Final products achieved by the biocatalytic hydrolysis of nucleic acids are oligonucleotide fragments of two to five base pairs. Moreover, S. marcescens endonuclease shows high stability, remaining active upon the addition of deleterious agents such as ionic, non-ionic, chaotropic agents, or denaturing compounds such as urea. Overall, those features strengthen its practical use for nucleic acid removal under many challenging processing conditions in a range of red biotechnology applications. A genetically engineered form of S. marcescens endonuclease was recombinantly produced using gram-negative Escherichia coli and made available years ago.

Based on its outstanding performance for nucleic acids removal, a new genetically engineered form of S. marcescens endonuclease called DENARASE® has been launched by the company c-LEcta GmbH (Leipzig, Germany). DENARASE® enzyme keeps the expected excellent broad operational window of pH and temperature of the endonuclease of S. marcescens. The patented production microorganism is not E. coli, but rather an endtoxin-free, gram-positive Bacillus sp. The fermentation media used are free of animal-derived feedstocks and antibiotics, so DENARASE® enzyme can be considered a BSE/TSE-free product with high viral safety (because no animal-derived materials are involved in its production). Also, DENARASE® enzyme does not display proteolytic activity, so it does not degrade peptides. That can be relevant when proteins are the marketable targets of the fermentation. DENARASE® enzyme can be delivered at high quantities at an excellent quality price ratio. And its manufacturing process is in full compliance with current good manufacturing practice (CGMP) requirements. Overall, that can open novel markets for endonucleases and trigger their use in already established applications, including medical and biopharmaceutical manufacturing, celltherapy, oncology, CAR T-cell development, and so on.

To illustrate the potential of DENARASE® enzyme for biopharmaceuticals, several successful case studies have been conducted. As stated above, one important application is the DNA/RNA removal from products made in fermentation processes that produce biopharmaceuticals, vaccines, or biological compounds following biosynthetic strategies. Along with a desired molecule, microbial cells are produced during fermentation, generating considerable amounts of nucleic acids (among other compounds). US FDA regulations restrict nucleic acid levels to <100 pg/dose (values applied to end-product samples) for biopharmacetical drug products before commercialization. DENARASE® enzyme can degrade nucleic acids rapidly and unselectively up to two to five base pairs. Thus manufacturers can comply with such regulations using DENARASE® enzyme under mild processing conditions.

Likewise, when microbial cells are disrupted, macromolecular nucleic acids can be released to culture medium. Performing cell disruption is a common downstream step during nonsecreted bioproduction (e.g., for adenovirus particles, antibodies, antigenic products, binding proteins, and vaccines). Released nucleic acids increase the viscosity of the fluid system significantly, thereby complicating subsequent processing units. To overcome such problems, DENARASE® enzyme can be incorporated to quantitatively hydrolyze host cell DNA and RNA and thus diminish liquid viscosity. Thus, process fluid handling (e.g., filtrations, chromatography) can be simplified significantly, thus reducing downstream costs to competitive levels. The endonuclease from S. marcescens displays a remarkably broad application range in biopharmaceutical production. This is attributable to its outstanding robustness, which enables application within an ample operational framework, keeping activity under a broad pH and temperature range and in the presence of deleterious chemical agents (e.g., urea). Under such conditions, DENARASE® enzyme keeps its unselective and excellent performance in the hydrolysis of nucleic acids of all types (DNA/RNA, single- and double-strand). 

DENARASE® enzyme is based on the recombinant expression in an endotoxinfree, gram-positive Bacillus strain as a microbial host, rendering a high purity of >99%. The patented fermentation procedure does not use animal-derived feedstocks, conferring the consideration of BSE/TSE-free product with high viral safety, in full compliance with European CGMP requirements. That enables DENARASE® enzyme to broaden the market opportunities for endonucleases, thus generating added value to biotechnological and biopharmaceutical applications such as nucleic acid removal, biosynthesis, cell therapy, oncology, CAR T-cell development, and so on. Interested? DENARASE® enzyme can be ordered directly from c-LEcta GmbH ( or through its new distribution partner VWR, part of Avantor in Europe (

In addition, a new enzymatic derivative called NuCLEANase® will be available from c-LEcta GmbH soon. It is the first nuclease with halal- and kosher-certified food-grade quality, thus envisioned for uses in the food industry.

Contact information: Pablo Domínguez de María is the chief executive officer of Sustainable Momentum, SL, a consultancy firm providing technical support in areas related to biorefineries, biotechnology, and sustainable chemistry.