Immobilized enzyme

Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include:

• Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.
• Economy: The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst. This is particularly useful in processes such as the production of Lactose Free Milk, as the milk can be drained from a container leaving the enzyme (Lactase) inside ready for the next batch.
• Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme.[3]

In the past, biological washing powders and detergents contained many proteases and lipases that broke down dirt. However, when the cleaning products contacted human skin, they created allergic reactions. This is why immobilization of enzymes are important, not just economically.

There are various ways by which one can immobilize an enzyme:

• Affinity-tag binding: Enzymes may be immobilized to a surface, e.g. in a porous material, using non-covalent or covalent Protein tags. This technology has been established for protein purification purposes. This technique is the generally applicable, and can be performed without prior enzyme purification with a pure preparation as the result. Porous glass and derivatives thereof are used, where the porous surface can be adapted in terms of hydrophobicity to suit the enzyme in question.[4]
• Adsorption on glass, alginate beads or matrix: Enzyme is attached to the outside of an inert material. In general, this method is the slowest among those listed here. As adsorption is not a chemical reaction, the active site of the immobilized enzyme may be blocked by the matrix or bead, greatly reducing the activity of the enzyme.
• Entrapment: The enzyme is trapped in insoluble beads or microspheres, such as calcium alginate beads. However, these insoluble substances hinder the arrival of the substrate, and the exit of products.
• Cross-linkage: Enzyme molecules are covalently bonded to each other to create a matrix consisting of almost only enzyme. The reaction ensures that the binding site does not cover the enzyme's active site, the activity of the enzyme is only affected by immobility. However, the inflexibility of the covalent bonds precludes the self-healing properties exhibited by chemoadsorbed self-assembled monolayers. Use of a spacer molecule like poly(ethylene glycol) helps reduce the steric hindrance by the substrate in this case.
• Covalent bond: The enzyme is bound covalentely to an insoluble support (such as silica gel or macroporous polymer beads with epoxide groups). This approach provides the strongest enzyme/support interaction, and so the lowest protein leakage during catalysis.[5]

Numerous enzymes of biotechnological importance have been immobilized on various supports (inorganic, organic, composite and nanomaterials) via random multipoint attachment. However, immobilization via random chemical modification results in a heterogeneous protein population where more than one side chains (amino, carboxyl, thiol etc) present in proteins are linked with the support with potential reduction in activity due to restriction of substrate access to the active site.[6]

In contrast, in site-directed enzyme immobilization, the support can be linked to a single specific amino acid (generally N- or C-termini) in a protein molecule away from the active-site. This way maximal enzyme activity is retained due to the free access of the substrate to the active-site. These strategies are mainly chemical but may additionally require genetic and enzymatic methods to generate functional groups (that are absent in protein) on the support and enzyme.

The choice of SDCM method depends on many factors, such as the type of enzyme (less stable psychrophilic, or more stable thermophilic homologue), pH stability of enzyme, the availability of N- or C-termini to the reagent, non-interference of the enzyme terminus with the enzyme activity, type of catalytic amino acid residue, the availability, price and the ease of preparation of reagents. For example, the generation of complementary clickable functionalities (alkyne

and azide) on the support and enzyme is one of the most convenient way for immobilizing enzymes via site-directed chemical modification.[7]

Another widely used application of the immobilization approach together with enzymes has been the enzymatic reactions on immobilized substrates. This approach facilitates the analysis of enzyme activities and mimics the performance of enzymes on e.g. cell walls.[8]