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This is a term which now covers a variety of methods of enzyme purification, the common factor of which is the more or less specific interaction between the enzyme and the immobilised ligand. In its most specific form, the immobilised ligand is a substrate or competitive inhibitor of the enzyme. Ideally it should be possible to purify an enzyme from a complex mixture in a single step and, indeed, purification factors of up to several thousand-fold have been achieved. An alternative, equally specific approach is to use an antibody to the enzyme as the ligand. Such specific matrices, though, are very expensive and cannot be generally employed on a large scale. Additionally, they often do not perform as well as might be expected due to non-specific binding effects. In general, affinity chromatography achieves a higher purification factor (with a median value in reported purifications of about ten fold) than ion-exchange chromatography (with a median performance of about three fold), in spite of it generally being used at a later stage in the purification when there is less purification possible.

A less specific approach, suitable for many enzymes, is to use analogues of coenzymes, such as NAD⁺, as the ligand. This method has been used successfully but has now been superceded by the employment of a series of water soluble dyes as ligands. These are much cheaper and, usually by trial and error, have been found to have surprising degrees of specificity for a wide range of enzymes. This dye-affinity chromatography was allegedly discovered by accident, certain enzymes being found to bind to the blue-dyed dextran used, as a molecular weight standard, to calibrate gel exclusion columns.

Another fortuitous discovery was hydrophobic interaction chromatography, found when it was noted that certain proteins were unexpectedly retained on affinity columns containing hydrophobic spacer arms. Hydrophobic adsorbents now available include octyl or phenyl groups. Hydrophobic interactions are strong at high solution ionic strength so samples need not be desalted before application to the adsorbent. Elution is achieved by changing the pH or ionic strength or by modifying the dielectric constant of the eluent using, for instance, ethanediol. A recent introduction is cellulose derivatised to introduce even more hydroxyl groups. This material (Whatman HB1) is designed to interact with proteins by hydrogen bonding. Samples are applied to the matrix in a concentrated (over 50% saturated, > 2M) solution of ammonium sulphate. Proteins are eluted by diluting the ammonium sulphate. This introduces more water which competes with protein for the hydrogen bonding sites. The selectivity of both of these methods is similar to that of fractional precipitation using ammonium sulphate but their resolution may be somewhat improved by their use in chromatographic columns rather than batchwise.

Careful choice of matrices for affinity chromatography is necessary. Particles should retain good flow and porosity properties after attachment of the ligands and should not be capable of the non-specific adsorption of proteins. Agarose beads fulfil these criteria and are readily available as ligand supports (see also Chapter 3). Affinity chromatography is not used extensively in the large-scale manufacture of enzymes, primarily because of cost. Doubtless as the relative costs of materials are lowered, and experience in handling these materials is gained, enzyme manufacturers will make increased use of these very powerful techniques.