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Removal of Nucleic Acids The purification of enzymes from bacterial homogenates and to a lesser extent animal tissue homogenates

is complicated by the presence of nucleic acids. Enzymes modified nucleic acid provide the foundation for the molecular biology technology. They are used to degrade, join or remove portions of the nucleic acids in a controlled and generally defined manner. These high molecular weight biopolymers increase the viscosity of the homogenate to an unmanageable level, at least in large scale operations where the next step in the process calls for filtration or centrifugation to remove cell debris. Some intracellular enzymes preparation contain nucleic which can give to increased viscosity interfering with enzymes purification procedures particularly ultrafiltration and centrifugation to remove cell debris. Factors such as shear, High pH, low ionic strength and the presence of endogeneous nucleases denatures nucleic acids. The problem of high viscosity may be eliminated by brief mechanical shearing in a Waring Blender, colloid mill or even vigorous mechanical stirring of the cell suspension. Nucleic acids can be precipitated by high molecular weight polyvalent cations such as protamine sulfate, cetyltrimethyl ammonium bromide (CTAB), streptomycin sulfate and polyethyleneimine. To remove Polyvalent cations form a complex between negatively charged Phosphate residues of the nucleic acid molecules and positively charged groups of the precipitant. The resulting complex is then removed by centrifugation. With extracts of E.coli it was shown that the effectives in precipating nucleic acids decreased in the order: POLYLYSINE> POLYETHYLENEIME> CTAB> STREPTOMYCIN PROTAMINE SULPHATE>MANGANESE CHLORIDE SULPHATE>

In the laboratory, Protamine sulfate has been used for the precipitation of nucleic acids from extracts of beef liver in the purification of glutamate dehydrogenase. After measuring the protein content, Protamine sulfate solution (20 mg/mL) at pH 7.0 was added to the partially purified enzyme solution to a final concentration of 100 mg/g protein. Approximately 60% of the nucleic acids were precipitated and removed by filtration. Subsequent purification of the enzyme by salt precipitation also reduced the nucleic acid content in the finished product. Welch and Scopes have purified yeast phosphofructokinase by adsorption of the enzyme to protamine-nucleic acid precipitates. Precipitation of proteins away from nucleic acids is possible with dextranPolyethylene glycol and high concentration of sodium chloride.

Other patented substances, which can be used for the precipitation of nucleic acids and some nonactive proteins from microbial enzyme extracts are water-soluble cationic synthetic polymers comprised of cationic monomers such as acrylamide, N-methyl-2aminoethylmethacrylate hydrochloride, 2-hydroxyethyl acrylate, and 2-(N,N, trimethylammonium)-ethyl methacrylate chloride. These synthetic polymers have been found useful in purifying enzyme extracts from a variety of microorganisms. A partial list includes, for example, M. flavins, B. megaterium, P. testeroni, and S. faecalis.