Reversible Covalent Immobilization of Enzymes via Disulfide Bonds

Abstract

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.

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

“…Covalent bonds provide powerful link between the lipase and its carrier matrix, allow its reuse more often than with other available immobilization methods [ 5 , 93 ] and prevent enzyme release into the reaction environment. [ 2 , 49 , 84 ] The method also increases half-life and thermal stability of enzymes when coupled with different supports like mesoporous silica, chitosan, etc.…”

An Overview of Techniques in Enzyme Immobilization

“…These thiol-containing enzymes are either in native forms or obtained through chemically modification or genetic engineering techniques, in order to provide them with reactive thiol groups. A detailed paper on immobilization of enzyme via their thiol group could be found here [33]. Alternatively, thiol containing enzymes can be immobilized onto supports, which fixed with reactive disulfides or disulfide oxides, through a thiolcontaining bifunctional linker which, on one end, forms disulfide bonds (S-S) to the surface, and on the other end, provides N-hydroxysuccinimide (NHS) groups that can react with the free amino groups on the enzyme.…”

Formulation of Laccase Nanobiocatalysts Based on Ionic and Covalent Interactions for the Enhanced Oxidation of Phenolic Compounds

“…The tradeoff analysis of the different outcomes will be critical to identify the most suitable option for its further use. Covalent bonding produces stronger bonds between the enzyme and the support, allowing its reuse more easily than with other available immobilization methods [36,37] and preventing the leaching of enzymes from the support [38,39]. In this study, different coatings as well as single-and multi-core nanoparticles were evaluated for laccase immobilization ( Table 2).…”