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Endotoxin Removal in Enzyme Purification

Creative Enzymes is specialized in providing the innovative and custom contract enzyme services satisfying the needs of clients from the pharmaceutical, biotechnological, and health care industry. Specifically, we offer the top-class development of enzyme purification in both small trial scales and large industrial scales from natural resources or production mixtures. With the addition of expertise from Creative Enzymes, your research is positioned to succeed.

Many recombinant enzymes are produced by the Gram-negative bacteria such as Escherichia coli. These products are quite often contaminated with endotoxins, lipopolysaccharides (LPS) present in the cell wall of most gram-negative bacteria. They are usually composed of three distinct regions: the O-antigen region, a core oligosaccharide, and Lipid A (Figure 1). The molecular mass of endotoxin is about 10-20kDa, depending mainly on the microbial strain. Enzymes must be as free as possible of endotoxins in order not to induce side effects when administered to animals or humans. However, endotoxins are very stable molecules, resisting to extreme temperatures and pH values in comparison to enzymes. Different processes have been developed for the removal of LPS from enzymes based on the unique molecular properties of the endotoxin molecules, including two-phase Triton X-114 extractions, ultrafiltration, hydrophobic interaction chromatography, ion exchange chromatography, and membrane adsorbers. These procedures offer different degrees of success in the separation of LPS from enzymes, depending on the properties of the enzyme of interest. 

Figure 1: Schematic view of the chemical structure of  endotoxin from E. coli O111:B4. Figure 1: Schematic view of the chemical structure of endotoxin from E. coli O111:B4.
Reference: Petsch D, Anspach F B. Journal of Biotechnology, 2000, 76(2): 97-119.

Endotoxin removal is one of the most difficult procedures in downstream processes during enzyme purification, given the labile chemical nature of enzymes. The form below lists most of techniques typically used to reduce endotoxin contamination of enzyme preparations.

Table 1: The techniques typically used to reduce endotoxin contamination of enzyme preparations.

Endotoxin Removal in Enzyme Purification

Creative Enzymes offers one-stop endotoxin removal service including precedent steps such as gene synthesis and enzyme expression, as well as the following endotoxin detection. The endotoxin removal service of Creative Enzymes is certificated to effectively reduce the endotoxin level to below 0.1 EU/mL, and the detection sensitivity is as high as 0.005 EU/ml. The resulted product meets strict requirements for downstream animal and cell experiments. With years of practices, Creative Enzymes works closely with clients through the purification process to solve technical challenges and mirror target processes. Creative Enzymes is your best choice for enzyme purification involving endotoxin removal.

Figure 2: The flow chart of endotoxin removal service. Figure 2: The flow chart of endotoxin removal service.



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