Dextrans, enzyme stabilization

In this article the preparation of one class of carbohydrate-enzyme conjugates, prepared by attachment of dextran to enzymes, is described in some detail and the properties of enzymes modified in this way are discussed. The molecular basis of enzyme stabilization by coupling with dextran is also considered. 

Our present approach in the preparation of new dextran-enzyme conjugates is initially to test the standard conditions. If such conditions do not give satisfactory results, either in terms of retention of activity or extent of conjugation, the effect of the three important parameters, pH, temperature and duration of reaction are then investigated to establish appropriate conditions for coupling. In some cases it has also been found useful to examine the effect of these variables on the stability characteristics and other properties of the resulting dextran-enzyme conjugates. 

In the presence of sucrose alone as the single substrate, initial reaction rates follow Michaelis-Menten kinetics up to 200 mM sucrose concentration, but the enzyme is inhibited by higher concentrations of substrate.30 The inhibitor constant for sucrose is 730 mM. This inhibition can be overcome by the addition of acceptors.31,32 The enzyme activity is significantly enhanced, and stabilized, by the presence of dextran, and by calcium ions. 

A number of enzymes to which soluble dextran had been attached after activation by cyanogen bromide have been characterized by Marshall and coworkers.7 All of the conjugated enzymes were found to be more stable than the native enzymes to heat, proteases, denatur-ants, and the absence of calcium. The attachment of dextran appeared to have stabilized the conformation of the enzyme. It was suggested that the attachment of the enzyme to the dextran polymer at several points fixed its conformation in much the same way as do intramolecular, disulfide bridges.7... 

Natural supports (agarose, dextran, cellulose, porous glass, silica, the optical fiber itself or alumina) and synthetic resins (acrylamide-based polymers, methacrylic acid-based polymers, maleic anhydride-based polymers, styrene-based polymers or nylon, to name a few) have been applied for covalent attachment of enzymes. These materials must display a high biocatalyst binding capacity (as the linearity and the limit of detection of the sensing layers will be influenced by this value), good mechanical and chemical stability, low cost, and ease of preparation. 

Alcohol oxidase could be immobilized onto DEAE-sepharose (Pharmacia) and had very high stability even when dried and rehydrated. We therefore tried to stabilize alcohol oxidize on a solid surface and as free enzyme using DEAE dextran. However this was not successful (Fig. 5). We also knew that lactitol had been used in stabilizing various antibodies during the manufacture of antibody based tests, however lactitol also had poor stabilizing properties when dried with alcohol oxidase (Fig 5). Unexpectedly, however, we found that a combination of DEAE dextran and lactitol gave excellent stabilization of die enzyme (Fig 5.). Further experiments showed that the combination of stabilizers was generic and would be used to stabilize broad selection of enzymes (Table 1 and see ref. 17). 

Figure 4. Effect of Dextrans on the Dry Stabilization of Alcohol Oxidase. Dextrans were added to the liquid enzyme at a concentration of 1-5%.

In addition to showing improved stability in the presence of protein denaturants, we have also found dextran-conjugated enzymes to show improved activity in the presence of such agents. For example, in the absence of calcium, native trypsin is inactive in 8M urea under the same conditions the modified enzyme displays 50% of the activity measured in the absence of urea (11). 

Miscellaneous. In addition to the effect of conjugation of a-amylase on its heat stability, stability in denaturants, and stability on cofactor removal, the attachment of dextran also results in improved stability of this acid-labile enzyme at pH values below about 5.0. Studies on the dependence of stability of Bacillus amyloliquefaaiens a-amylase on pH showed that the conjugated enzyme retained 20, 15 and 7% more activity at pH 3.5, 4.0 and 4.5 than did the native enzyme. 

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