Enzyme immobilization is the process of attaching or confining enzymes to a solid support or within a matrix so that they retain their catalytic activity and can be reused multiple times. Unlike free enzymes which are dissolved in a solution, immobilized enzymes are fixed in on solid support or matrix place, which gives practical advantages in industrial and laboratory applications.
The classification of the process of enzyme immobilization is divided mainly in two category which based on the confinement of enzyme either on the surface or inside the support matrix. Ech class is again further classified as given below:
Covalent coupling involves the formation of strong covalent bonds between functional groups on the enzyme and reactive groups on a solid support material, leading to permanent attachment of the enzyme on support material.
Common support material| Functional Groups on Enzymes | Functional Groups on Support material |
|---|---|
| –NH₂ (from lysine) | Hydroxyl group on Polyacrylamide, Polyvinyl alcohols |
| –SH (from cysteine) | Aldehyde and Acetal group of Polymers |
| –COOH (from glutamate/aspartate) | Amide group of Nylon |
| –OH (from tyrosine) | Amino and related group of Silica gel |
| Imidazole group (on Histidine) | Carboxylic acid group of glutamic acid, carboxy methyl cellulose |
In this process coupling agents are used. Coupling agents are chemical linkers used to facilitate the covalent bonding between the enzyme and the support material. Their main roles of coupling agents is to activate the support material and formation of active binding group, this modify the surface of support matrix, so that it can react with functional groups present on the enzyme (–NH₂, –COOH, –SH), and form stable covalent bonds to ensure strong attachment that resists leaching or dissociation of enzyme from support matrix.
Coupling Agents| Coupling Agent | Mechanism | Example |
|---|---|---|
| Glutaraldehyde | Forms Schiff base (imine) bonds between aldehyde and enzyme –NH₂ groups | Immobilization of glucose oxidase on silica beads |
| Cyanogen bromide (CNBr) | Activates –OH groups to react with enzyme –NH₂ groups | Immobilization of protease on CNBr-activated Sepharose |
Adsorption is the simplest and most straightforward method. It involves the physical binding of enzyme molecules onto the surface of an inert and insoluble carrier material without forming covalent bonds. In this method, the enzymes adhere to the surface of the carrier via weak, non-covalent interactions, such as van der Waals forces, hydrogen bonding, hydrophobic interactions, or electrostatic (ionic) attractions. Because these interactions do not alter the enzyme's active site or structure significantly, the enzyme typically retains much of its native activity after immobilization.
The support material which are used for adsorption methods, over which the enzymes are getting adsorbed areStatic pore adsorption is a specific type of physical adsorption where enzymes are immobilized by diffusing into and adhering to the internal pores of a porous support material. It is called “static” because adsorption of enzyme over solid support material is achieved by placing the solid support material in enzyme solution, and the enzyme are adsorbed on solid surface by simple diffusion and no physical force or agitation is involved in this.
The complexation method (also called chelation or metal ion affinity binding) involves immobilizing enzymes through the formation of coordination complexes between metal ions and functional groups on the enzyme.
It relies on specific and reversible interactions between metal ions (like Ni²⁺, Cu²⁺, Zn²⁺) and electron-donating groups such as imidazole, carboxyl, thiol, or amino groups present on enzymes.
In complexation-based immobilization, enzymes bind to a support material via coordination bonds involving transition metal ions. These metal ions form complexes with electron-donating groups present on both the enzyme surface (like –NH₂, –COOH, –SH, imidazole groups) and the carrier matrix. Metal ions which are used in this method (like Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺, Fe³⁺) are used as linkers. Whereas Common supports include agarose, sepharose, silica beads, etc., modified with chelating agents are used in this method.
In the first step of the process, the support is loaded with a metal ion. Then the enzyme binds to the immobilized metal ion (which was attached on the support material in first step) through specific donor atoms of enzyme (like N in histidine, S in cysteine). In this way a Coordination complex forms as support – metal ion – enzyme group, and ultimately fixing the enzyme to the support.The immobilization of enzymes in chelation method achieved by forming a special type of bond between metal ions and certain parts of the enzyme surface. In this method, a chelating agent (like IDA or NTA) is first attached to a solid support material Agarose Beads (e.g., Sepharose), Silica or Glass Beads, Polymers (e.g., Polystyrene, Polyacrylamide), Cellulose etc.
This chelating agent, which is attached over support material, then treated with any metal ion such as Ni²⁺, Co²⁺, Zn²⁺, or Cu²⁺ to form metal-chelate complexes. The enzyme is added to this metal-chelate complex. due to this, the metal ion can then bind to specific parts of the enzyme—which have electron-rich groups. This type of bond is not permanent (not covalent), but rather a reversible connection, which means the enzyme can be attached or removed by changing the conditions.Crosslinking is a method of enzyme immobilization in which enzyme molecules are chemically bonded to each other using special chemicals called multifunctional reagents. This creates a stable, three-dimensional network of enzyme molecules.
In this method, special chemicals called multifunctional reagents were used. These chemicals have two or more reactive groups, which allow them to form covalent bonds (very strong chemical links) between enzyme molecules. The enzyme which is subjected to immobilize is treated with a multifunctional reagent. These reagents react with certain functional groups (like –NH₂ groups) on the enzyme surface and form covalent bonds between enzyme molecules. This reaction joins the enzymes together by forming covalent bonds, thus forms insoluble 3D aggregates or networks of enzymes—like a mesh—without needing any solid support. Most commonly used crosslinking agents are Glutaraldehyde, Toluene diisocyanate, Dextran polyaldehyde etc.Entrapment by occlusion is a passive immobilization technique where molecules are physically trapped within the pores or voids of a crosslinked gel matrix during or after gel formation. There is no chemical bonding between the entrapped substance and the gel network.
A three-dimensional polymer structure was generated by using alginate, polyacrylamide, gelatin, PEG etc. This process takes place during or immediately after the formation of the gel, as the polymer chains crosslinked to each other and form a three-dimensional mesh-like structure, the target molecules (enzyme) become “occluded” (i.e., trapped or enclosed) within the voids, pores, or interstitial spaces of the network. Calcium alginate beads are one of the most widely used systems for entrapment by occlusion.Encapsulation in Microcapsule Enzyme Immobilization is a technique where enzymes are enclosed within semi-permeable membranes (microcapsules), allowing the exchange of substrates and products while keeping the enzyme retained inside. The Semi-permeable membrane allows diffusion of small substrates/product but prevents enzyme loss and denaturation.
Microcapsules are mostly spherical with diameters usually in the micro- to millimetre range and exhibit core-shell structures of variable complexity. Liposomes and vesicles formed from amphiphilic (co)-polymers (polymersomes) are widely used to prepare membrane-coated microcapsules. Hydrogels, sol–gels and other organic–inorganic hybrid materials, or layer-by-layer structures made through controlled assembly of polyelectrolytes are used to prepare microcapsules composed of internal polymeric networks.