## 5 Innovative Approaches for Enzyme Immobilization
### Nanomaterial-Based Enzyme immobilization
#### 5.1.3 Magnetic Nanoparticles (MNP)-Based Enzyme Immobilization  
Magnetic nanoparticles (MNPs), composed of iron, nickel, aluminum, and cobalt, are widely used due to their magnetic responsiveness [122]. Their large surface area, superparamagnetic properties, and adaptability make them ideal for industrial applications, while they also enhance enzyme activity and prevent denaturation, especially when encapsulated in biocompatible materials like polydopamine, chitosan, or silica [123]. Immobilized metal affinity magnetic nanotechnology (IMAN), based on IMAC, employs MNPs as carriers to chelate metal ions (e.g., Cu\({}^{2+}\), Ni\({}^{2+}\), Zn\({}^{2+}\), Co\({}^{2+}\)) and bind proteases, maintaining enzyme activity capacity [124].  
Magnetite (Fe\({}_{3}\)O\({}_{4}\)) is the most widely used MNP for enzyme immobilization due to its low toxicity, high magnetic susceptibility, large surface area, biocompatibility, and superparamagnetic properties at ambient temperatures [9]. Studies show that chitosan-coated MNPs retain 80% of their enzyme activity after 30 days at 4 \({}^{\circ}\)C and 48.8% after 13 cycles, yielding 64.45% glucose (21.84 g/L) [125], while glucosidase immobilized on MNPs achieved a 93% binding efficiency and maintained 50% of its activity after 16 cycles [126].  
It has been reported that xylanase and lacase can be immobilized on magnetic supports such as chitosan beads with Fe\({}_{3}\)O\({}_{4}\) nanoparticles, magnetic graphene oxide (GO-MNP), bioanodes with metals, and Fe\({}_{3}\)O\({}_{4}\)@NC [127; 128]. These immobilized biocatalysts enhanced the efficiency, reusability, and storage stability for applications in cellulose/Xylan hydrolysis and phenolic compound degradation.  
Reports indicate that MNPs such as magnetic Janus silica nanoparticles and Fe\({}_{3}\)O\({}_{4}\) MNPs effectively immobilized protease, and cellulase exhibited stability under varying temperature and pH levels [129; 130]. These immobilized enzymes showed potential for dye decolorization and juice clarification. MNPs-based immobilization offers advantages like high surface area, stability, low toxicity, and biocompatibility [131]. Cross-linking agents (e.g., glutaraldehyde) enhance enzyme thermostability by reducing steric hindrance and increasing flexibility [131]. Additionally, coating MNPs with organic and inorganic compounds can reduce toxicity, broadening their application potential.  
In summary, MNP immobilization enhances enzyme stability and catalytic efficiency by optimizing active site orientation, minimizing diffusion barriers, and improving substrate accessibility [132]. It also simplifies enzyme separation and recycling, lowering costs for industrial and environmental applications.  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Enzyme** & **MOF** & \begin{tabular}{c} **Immobilization** \\ **Method** \\ \end{tabular} & **Enzyme Reaction** & **Improved Enzyme Application** & **Ref.** \\ \hline \multirow{4}{*}{\
\begin{tabular}{c} Horseradish \\ peroxidase lysozyme \\ \end{tabular} } & \multirow{4}{*}{UnO-66-F4} & \multirow{4}{*}{Encapsulation} &
\begin{tabular}{c} 3,3\({}^{\prime}\)5,5\({}^{\prime}\) tetramethylbenzidine (TMB) \\ used as the substrate and H\({}_{2}\)O\({}_{2}\) as an oxidizing agent in the presence of 1-phenyl-ethanol with vinyl acetate \\  & & & & Improved activity and stability, \\  & & & & enhanced temperature tolerance over a wide range. \\  & & & & 1-phenyl-ethanol with vinyl acetate \\  & & & & & Not exposed to digestive enzyme pepsin or long-term \\  & & & & & storage, providing protection from harsh conditions. \\  & & & & &