Covalent immobilization: A review from an enzyme perspective

Enzymes are indispensable in biotechnology, serving as biological catalysts in applications across various domains, including biosensors, fine chemicals production, pharmaceuticals, and drug development. In this context, enzyme immobilization is crucial for ensuring their retention on devices while preserving their activity over extended periods. Various immobilization methods, both physical (adsorption, affinity bonding, entrapment, encapsulation, and ionic bonding) and chemical (covalent bonding and crosslinking), have been explored, each exerting distinct impacts on enzyme stability and activity. Among these, chemical immobilization typically offers superior stability compared to physical methods due to the formation of stronger bonds between the enzyme and the support material. Covalent immobilization is commonly used due to its efficacy in enhancing enzyme stability. Carbodiimide chemistry and Schiff base reactions are the two most common covalent bond techniques used for immobilization, owing to the functional groups involved in the reaction (–NH2 and –COOH), which are commonly found on enzymes surface. This review provides a background on enzymes and the various methods for immobilizing them onto materials, before delving into carbodiimide and Shiff base reaction techniques. The characteristics, advantages, and disadvantages of both these techniques are discussed, including bond formation, reaction condition, and implications for application. Additionally, the review underscores the significance of enzyme orientation, structure, and conformational changes. Achieving optimal orientation and minimizing conformational alterations are critical factors in developing a stable, highly active, selective, scalable, and reproducible enzymatic biosensor.