Proteinases detergents

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Sources of Proteinases. More than 80 per cent of all industrial enzymes are hydrolytic in action and most are used for the depolymerization of natural substances. Almost 60 per cent of these enzymes are proteolytic, and used by the detergent, dairy and leather industries. 

Radiolabeled (3H, l4C, 35S, or l25I) protein or cell extract prepared in PBSA containing 10 3M phenyl methyl sulfonyl fluoride (PMSF) as proteinase inhibitor, and 0 5-1.0% nonionic detergent... 

In contrast to tissues, tissue culture cells are readily lysed with detergent [11]. Adherent cells from a culture plate are first scraped using a rubber policeman into a small volume of phosphate-buffered saline (137 mMNaCl, 2.7 mM KC1, 4.3 mM Na2HP04, 1.4 mM KH2P04, pH 7.3) and harvested by centrifugation at 1500 x g for 10 minutes at 4°C. The cellular pellet is resuspended in ice-cold TE so that 1 mL contains 100 million cells. After the addition of 10 volumes of freshly prepared digestion buffer (10 mM Tris-Cl, pH 8, 0.05 EDTA, pH 8, 0.5% Sarcosyl, and 100 pg/mL proteinase K), the sample is incubated at 50°C for 3 hours. DNA is recovered by ethanol precipitation after extraction with phenol, phenol-chloroform, and chloroform as described earlier. 

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by... 

Fast and simple methods for extraction of DNA or RNA from cells, whole blood, and other fluids have been reviewed by Bloch24 and Kawasaki.25 In general, target DNA is released from the cells or virus by proteinase K digestion in the presence of detergent followed by heat inactivation of the proteinase K. A small aliquot of the cell lysate is then used directly as the template in the amplification reaction. For cell culture supernatant fractions, the proviral DNA released from the lysed cells can be directly... 

Enzymes had been used in detergent powders in the U.S. and Europe as early as 1960. They were subsequently withdrawn in the U.S., but not in Europe, when the raw proteinase used at the time proved to have an adverse effect on the health of detergent plant workers. Improvements in the enzymes, specifically encapsulation, eliminated their dustiness and made it possible to use these materials in detergent plants without adverse health effects. 

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