Reversible covalent immobilization of enzymes via disulfide bonds.
This enzyme immobilization approach involves the formation of disulfide (-S-S-) bonds with the support. Thus, enzymes bearing exposed nonessential thiol (SH) groups can be immobilized onto thiol-reactive supports provided with reactive disulfides or disulfide oxides under mild conditions. The great potential advantage of this approach is the reversibility of the bonds formed between the activated solid phase and the thiol-enzyme, because the bound protein can be released with an excess of a low-molecular-weight thiol (e.g., dithiothreitol [DTT]). This is of particular interest when the enzyme degrades much faster than the adsorbent, which can be reloaded afterwards. 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. Disulfide oxides (thiolsulfinate or thiolsulfonate groups) can be introduced onto a wide variety of support materials with different degrees of porosity and with different mechanical resistances. Procedures are given for the preparation of thiol-activated 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.
Cátedra de Bioquímica, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Uruguay.
Karen
K
Ovsejevi
Carmen
C
Manta
Francisco
F
Batista-Viera
D000327:Adsorption; D003432:Cross-Linking Reagents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D057075:Enzyme Assays; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D019163:Reducing Agents / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry; D017186:Titrimetry; D001616:beta-Galactosidase / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D019163:Reducing Agents; C037851:thiol-sepharose; D012685:Sepharose; D001616:beta-Galactosidase; D004229:Dithiothreitol