Tunable Polymeric Scaffolds for Enzyme Immobilization

Abstract

The number of methodologies for the immobilization of enzymes using polymeric supports is continuously growing due to the developments in the fields of biotechnology, polymer chemistry, and nanotechnology in the last years. Despite being excellent catalysts, enzymes are very sensitive molecules and can undergo denaturation beyond their natural environment. For overcoming this issue, polymer chemistry offers a wealth of opportunities for the successful combination of enzymes with versatile natural or synthetic polymers. The fabrication of functional, stable, and robust biocatalytic hybrid materials (nanoparticles, capsules, hydrogels, or films) has been proven advantageous for several applications such as biomedicine, organic synthesis, biosensing, and bioremediation. In this review, supported with recent examples of enzyme-protein hybrids, we provide an overview of the methods used to combine both macromolecules, as well as the future directions and the main challenges that are currently being tackled in this field.

Keywords: biocatalysis; enzyme immobilization; enzyme-polymer hybrids; nanocarriers; polymeric supports; stabilization of enzymes.

SCHEME 1

The synthesis of enzyme-polymer hybrids requires the selection of a rationally designed methodology in line with the selected polymeric material and the properties of the enzyme, which should keep the catalytic performance all along the synthesis procedure and in the eventual supramolecular structure. In this process, several parameters (some of which are highlighted in the scheme) need to be considered for a successful fabrication of enzyme-polymer hybrids.

FIGURE 1

Schematic illustration of polymer-enzyme hybrids synthesized through different covalent-based methodologies. Strategies (A–C) require the synthesis of a preformed polymer (grafting-to approach) and (D) refers to the in situ synthesis of the enzyme-polymer hybrid (grafting-from approach). (A) Non-specific grafting of proteins, using polymers with a single reacting group and proteins with several target residues. (B) Multi-point strategy using polymers with multiple anchoring sites. (C) Site-specific grafting using a biorthogonal approach. (D) Grafting-from approach that entails the conjugation of the macroinitiator to the protein and the subsequent in situ polymerization after the addition of the monomers.

FIGURE 2

Polymers can be displayed over the enzyme surface in different configurations according to the grafting density. So called “mushroom” or “brush” configurations are achieved at low and high grafting density, respectively.

FIGURE 3

Schematic illustration of polymer-enzyme hybrids synthesized through different non-covalent based methodologies. (A) Adsorption of polymers on the surface of proteins. (B) Ionic interaction-based enzyme-polymer hybrids. (C) Entrapment of enzymes in the porous network of polymers. (D) Affinity-driven formation of enzyme-polymer hybrids.

FIGURE 4

Synthesis of single enzyme nanogels. (A) Fabrication procedure of single enzyme nanogels by in situ radical polymerization. (B) We showcase some of the most used monomers and crosslinkers. Selected monomers can provide hydrophilicity (red), the ability to complex metal cations (purple) or tune the ionic interactions within the polymeric shell (blue). Moreover, the use of labile crosslinkers allows the release of the protein under external stimuli (presence of a peptidase, light or redox reagents for the examples drawn in green).

FIGURE 5

Assembled enzyme-polymer supramolecular hybrids in which the enzyme is displayed to the environment. (A) Enzyme-polymer micelles. (B) Dendrimer-enzyme hybrids. (C) Organic-inorganic enzyme hybrids. (D) Giant amphiphile structures.

FIGURE 6

Assembled enzyme-polymer supramolecular hybrids in which the enzyme is confined in the inner cavity of the structures. (A) Reverse micelle-enzyme hybrid. (B) Polymersome-enzyme hybrid. (C) PICsome-enzyme hybrid.

FIGURE 7

Enzyme-MOF hybrids can be synthesized either by the infiltration of the protein inside the pores of the material (A) or through the immobilization of the enzyme on the surface of the organic framework (B).

FIGURE 8

Schematic illustration of enzyme-polymer microhybrids described in this work. (A) Enzyme-hydrogel microparticles. (B) Microparticles or vesicles formed by LbL assembly. (C) CLEAs or MOEAs. (D) Enzyme-polymer fiber hybrids.

FIGURE 9

Schematic illustration of enzyme-polymer monoliths and enzyme-polymer film types formed following different approaches mentioned in this work: (A) polymer monoliths; (B) assembly of SENs; (C) assembly of MOFs; (D) assembly of microparticles; and (E) attachment to polymer brushes.