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Solubility-Based Enzyme Purification

Creative Enzymes is a worldwide leader specialized in enzyme services. For years, Creative Enzymes has helped customers with multidisciplinary specialties and in-depth scientific support in enzyme expression and purification. The combination of several enzymology areas created the robust platform of Creative Enzymes for new technology and quick development in enzyme purification. We are proud to provide enzyme purification services with high flexibility to precisely meet the need of our diverse and unique customers.

Generally, protein mixtures are subjected to a series of separations, each based on a different property to yield a pure protein. Thus, a variety of purification techniques are available. Solubility-based purification technique is a kind of method which changes the solubility of protein to separate it from other ingredients. Virtually, there are many external conditions that change the protein solubility, such as the pH of the solution, ionic strength, dielectric constant and temperature. However, under the same conditions, different proteins have their own different solubility because of their molecular structures. According to the characteristics of protein molecular structures, appropriate changes in these external conditions can selectively control the solubility of one component of the protein mixture, achieving the purpose of separation and purification of proteins ultimately. Solubility-based purification methods commonly used include: isoelectric point precipitation, pH adjustment, protein salting in and salting out, organic solvent method, double water phase extraction method, and reverse micelle extraction method. Creative Enzymes is able to use these methods in combinations to give the target enzymes at the desired yield and purity.

When salt ions are added, the charge of protein molecules around will be increased, then the interaction between protein and solvent molecules will be promoted, and finally the protein solubility will increase, this phenomenon is called salting in. On the contrary, when the salt concentration continues to increase, a large number of salt ions reduce the relative concentration of water, then the hydration of protein will be weakened, and finally the proteins aggregates and precipitates, this phenomenon is called salting out. These two effects are different for different proteins. Fractionate by raising the “salt” just below point where protein to be purified becomes insoluble, removed other precipitated proteins by centrifugation, then raise the “salt” to precipitate protein and collect it by centrifugation.

Proteins can be either positively (cation) or negatively (anion) charged based on pH conditions. The characteristic pH of a solution at which the net charge on protein is zero (positive and negative charges are equal) is defined as the isoelectric point (pH). The isoelectric point of a protein is an important property because it is at this point that the protein is least soluble. Different proteins have different isoelectric points, so one can manipulate the relative solubility of a mixture of proteins by changing the pH. Actually, the method of changing the solubility of the enzyme by using the temperature changes is the same as the pH changes.

Solubility-Based Enzyme Purification Figure: Solubility-based purification method. (a) mixture of three protein-white, grey, black; (b) solution altered: black protein precipitates (supernatant removed); (c) solution altered again: grey protein precipitate (white protein remains in supernatant).

The addition of a water miscible organic solvent can significantly reduce the dielectric constant of the solution, so that the electrostatic interaction between the enzyme molecules is enhanced and the enzymes will precipitate. Organic solvents commonly used are methanol, ethanol, and acetone. Therefore, this method can selectively precipitate some enzymes by increasing the concentration of water miscible organic solvents.

Generally, the solubility-based enzyme purification technique is the first step in an enzyme purification typically. And this method is often simple-operated compared with other purification methods. However, it also has some disadvantages. For example, enzymes purified by “salting out” often need to be further purified to remove the high concentrations of ions. Sometimes the changes of pH and temperature may destroy the enzyme activity, or even cause the inactivation of the enzyme. Depending on extensive research experience on enzyme purification and equipped with the cutting-edge instruments, Creative Enzymes is able to design the purification process to minimize the impact of these disadvantages. We provide the most reliable and multiple enzyme purification services to solve any specific problems which may lay on your way to enzyme applications. Our customer-oriented and comprehensive purification service is your first choice in enzyme purification.

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