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aBecause supports have quite different swelling factors in water, it is sometimes more useful to know the binding capacity in |imol of GSH per g of swollen and suction-dried (wet) derivative.

aBecause supports have quite different swelling factors in water, it is sometimes more useful to know the binding capacity in |imol of GSH per g of swollen and suction-dried (wet) derivative.

for immobilization. Thus, genetically engineered glucose dehydrogenase has been immobilized on a Thiopropyl-Sepharose column (15).

After enzyme immobilization onto TS/TSI-gels, the remaining reactive groups in the matrix could react with hidden thiol groups in the protein, leading to an activity loss, specifically during thermal treatments. In order to avoid this effect, excess reactive structures can be blocked by coupling polar low-MW thiols. Thus, a P-galactosidase derivative blocked with glutathione retained 100% of initial activity after 1 h at 50°C, whereas the unblocked derivative only retained 16% of the activity (10).

On the other hand, different nanoenvironments can be generated by reacting the excess of gel-bound disulfide oxides moieties with apolar, polar, anionic, or cat-ionic thiols. In that way, it might be possible to tailor the matrix surface so as to maximize enzyme stability in particular applications (10,16).

In conclusion, reversible covalent immobilization of enzymes through thiol-disulfide exchange reactions has the following advantages.

• It involves thiol groups, which are generally the most reactive groups found in proteins, because of the high nucleophilicity of the corresponding thiolate ions.

• Because thiolate ions exist at reasonable concentrations at neutral to weakly alkaline pH values, it is possible to perform enzyme immobilization under mild conditions and at relative high rates.

• These immobilization methods are absolutely specific for thiols.

• Resulting from the fact that thiol groups are scarce in proteins and located in specific regions, the enzyme can be immobilized in an oriented way, exposing the active site.

• Immobilization methods based on thiol-disulfide exchange reactions are unique, because they combine a very stable covalent attachment with the possibility of releasing the bound material. Thus, after enzyme inactivation, the immobilized material can be easily released by reduction.

• Disulfide oxides can be introduced onto a wide variety of support materials with different degrees of porosity and with different mechanical resistances that make them suitable for many analytical and preparative applications.

• The activated supports show high storage stability because thiol-reactive groups remain unchanged after 2 yr at 4°C.

• Sulfur content makes these gels highly resistant to microbial growth, so it is not necessary to use sodium azide as a preservative.

• The activated gel structures are completely regenerable for TSI- and PyS2-gels, and 50% regenerable for TS-gels.

• The possibility of reusing the polymeric support after inactivation of the enzyme may be of interest for the practical use of immobilized enzymes in large-scale processes in industry, where their use has often been hampered by the high cost of the support material.

In Subheading 3., procedures are given for the preparation of thiol-reactive solid phases and the covalent attachment of thiol-enzymes to the support material via disulfide bonds. The possibility of reusing the polymeric support is also shown after reductive detachment, chemical reactivation, and reattachment of fresh thiol protein.

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