Detergent-enzymatic method

Neutral monosaccharides, uronic acids, hexosa-mines, and sialic (neurominic acids) are identified and determined by specific colorimetric reactions. The principle behind the techniques rests on the condensation of the degraded products of the neutral monosaccharides (hexose, pentose and methyl pentose) by sulfuric acid with anthrone, cysteine hydrochloride, orcinol, and phenol reagents. Uronic acids may be determined by colorimetric and manometric procedures. While sialic acids are determined after chemical/enzymatic hydrolysis, gravimetric and Van-Soest detergent based methods are used to determine cellulose, hemicellulose, and fiber. 

In a general procedure, after the cell wall is broken by mechanical or enzymatic methods (lysozyme), the resulting cell sap is treated with a protein-denaturing agent, such as phenol, or a detergent (dodecyl sulfate, lauryl sulfate), which precipitates proteins. Several extractions are frequently necessary. The final nucleic acid solution is treated with ethanol, to precipitate nucleic acids, or dialyzed against a suitable buffer solution. A review by Kirby (14) discusses the various isolation procedures used and their advantages and inconveniences. 

Enzymatic Gravimetric Methods for TDF, SDF, and IDF. These methods use an a-amylase and protease to remove starch and reduce protein. They differ from each other in the conditions for gelatinization of starch. Elimination of detergent permits recovery of soluble fiber, which is not possible with the detergent methods. 

Chemical lysis, or solubilization of the cell wall, is typically carried out using detergents such as Triton X-100, or the chaotropes urea, and guanidine hydrochloride. This approach does have the disadvantage that it can lead to some denaturation or degradation of the produci. While favored for laboratory cell disruption, these methods are not typically used at the larger scales. Enzymatic destruction of the cell walls is also possible, and as more economical routes to the development of appropriate enzymes are developed, this approach could find industrial application. Again, the removal of these additives is an issue. 

Once purified, DNA is a fairly stable polymer if stored appropriately. Since living cells contain many other complex biomolecules besides DNA, methods exist that allow the isolation of DNA in pure form. More details on this topic are presented in Chapter 8. Routine methods of DNA isolation in solution, however, cause some unavoidable shearing of DNA due to hydro-dynamic shear forces, and as a result, the average size of isolated DNA is about 100 to 200 kilobases (kb). The basic steps in DNA isolation involve cell disruption and lysis by treatment with detergents, removal of cellular proteins by either enzymatic digestion with a protease or extraction with... 

Cell lysis Mechanical methods pressure shearing, ultrasonic disintegration, bead-mill homogenizers Nonmechanical methods enzymatic lysis, osmotic lysis, freezing and thawing, detergent-based lysis and electroporation... 

When the protein is present within a cellular organelle, these methods can still be suitable. However, they may be preceded by isolation of the organelles. Sometimes, the protein has low solubility in the extraction medium, and produces a particulate system requiring specific techniques. These proteins can be extracted through thermal, chemical, or enzymatic treatments, and in some cases detergents are needed for solubilization. In any case, it is essential that a suitable solvent is selected for protein extraction. 

The use of microsomes along with UDPGA as a cofactor assay to measure UGT enzyme activity has been hampered historically by the fact that this enzymatic activity in microsomes is often in a latent form and requires activation by physical or detergent-induced disruption of the membrane matrices. Recently, a generic method involving the addition of the pore-forming peptide alamethicin to overcome the latency exhibited by this enzyme system has been described.99 The inclusion of alamethicin seems to provide a more consistent method of assessing UGT enzyme activity. 

Oberholzer, T. et al.. Enzymatic reactions in liposomes using the detergent-induced loading method, Biochim. Biophys. Acta, 1416, 57-68, 1990. 

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