How can enzymes be selectively purified with the help of this technique?
To apply this purification technique, the enzyme of interest must have affinity to detergents as Triton X-114. In this case, the detergent is added at an appreciable concentration (4-6%) in order to form mixed micelles with the enzyme of interest. In some cases, the enzyme is initially extracted from cells with detergent. Then the medium containing detergent-enzyme is heated to induce a phase separation (cloud point) at a relatively high temperature (around 40°C). Two aqueous phases is consequently formed: aqueous phase (containing units of the detergent and impurities) and a detergent-rich phase containing the enzyme. A centrifugation is required to separate these phases and to collect the detergent-rich phase which deposits. This enzyme-containing phase is diluted and treated to collect the enzyme .
An alternative two phase system is one using a high molecular weight nonionic polymer such as polyethylene glycol (PEG) and a concentrated salt solution (typically phosphate). Hydrophobic enzymes migrate to the PEG layer and hydrophilic ones to the salt layer. There were plenty of papers in the 1990-2000 era on this method. Biggest problem with this method is the lack of predictability in where your enzyme will end up. Having tried the method, it does work, but not as a single step purification. There are additional papers on modifying the PEG to make it an affinity chromatography extractant.
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