## 5 Innovative Approaches for Enzyme Immobilization
### Integration of Synthetic Biology and Immobilization  
Enzyme immobilization improves traditional covalent immobilization by preventing random binding, thus safeguarding active sites. Strong chemical bonds reduce enzyme leakage and contamination [198]. However, only enzymes with reactive amino acids (e.g., Thr, Cys, and Lys) are suitable [199]. Figure 6: Different types of enzyme-immobilized microfluidics. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Enzyme** & **Hydrogel** & **Improved Enzyme Activity** & **Application** & **Refs. ** \\ \hline \multirow{2}{*}{Xylanase} & Superabsorbent hydrogels (SHs); carboxymethyl cellulose-\(\mathbf{g}\)-poly & Enhanced hydrolysis performance and reusability & Hydrolysis of pulp paper to release & [200] \\  & (acrylic acid-co-acrylamide) hydrogel & Improved the bleaching of paper pulp. & Improved the & \\  & Bacterial cellulose (BC)-chitosan & Highest thermal and operational stability and & Hydrolysis of p-nitrophenyl butyrate to & \\  & composite hydrogel & improved enzyme half-lite time. & Hydro -nitrophenyl & \\  & Polyvinyl alcohol/sodium alginate & Noticeable improvement in storage stability, & Catalytic \(\beta\)-nitrophenyl & \\  & (PVA/Alg) hydrogel & Imitation of enzyme leakage, improved enzyme & & \\  & & affinity to the substrate, and catalytic efficiency. & & \\  & & & \\  & Poly (2-hydroxyethyl & Immobilized enzymes, maintaining over & \\  & methacrylate)-calcium magnesium & 80\% catalytic activity after one month, with & \\  & carbonate & superior stability. & & \\  & (pHEMA-CaMg(CO))\({}_{2}\)) hydrogel & Enzyme was reused 15 times and retained its & & \\  & & & 70\% activity, meanwhile it showed 60\% activity & \\ \(\beta\)-mannanase & Sodium alginate-grafted-\(\beta\)-cyclodextrin & after 30 days of storage at 4 °C. & Enzymatic catalysis of locust bean gum (LBG) & \\  & & Increased the thermostability and half-life of the & to produce mannose sugar. & \\  & & enzyme and reduced activation energy. & & \\  & Poly (acrylamide/vinylsulfonic acid) & Maintained their stability over a wide pH and & \\  & PA/VSA and poly (acrylamide) & temperature range, with storage stability, after & \\  & PA hydrogels, & successive batch reaction. & & \\  & carboxymethyl cellulose grafted & Enzyme retained nearly 60\% of its maximum & \\  & copolymers of 2-acrylamide-2 methyl & activity at 90 °C, with storage stability. & & \\  & & & \\  & (CMC-g-poly & Enzyme showed 154.8\% increase in conversion of & \\  & (AMPS-co-A\(\Delta\)Am)) hydrogels & & alkaline-treated sugar beet pulp. & \\  
Despite benefits like stability and reusability, challenges remain, including short half-lives, dependance on non-natural substrates, and decreased activity under harsh conditions. In situ immobilization, combining bioengineering and synthetic biology, offers a promising solution by enhancing the enzymes and carrier materials. Enzyme engineering further optimizes stability, activity, and resistance using protein engineering and recombinant DNA technology [208]. Advances in enzyme engineering, including directed evolution, metagenomics, rational design, and chemical modification, have optimized catalytic activity by targeting binding and active sites. Multi enzyme catalytic cascades further boost biosynthesis and biotransformation. For example, engineered xylanases exhibit improved thermal stability for use in animal feed and food [209]. Immobilization and site-directed mutagenesis enhance stability and enable controlled enzyme function [210]. A notable example is the engineering of 41 NOV1 dioxygenase variants, which achieved tenfold higher activity and greater stability than the native enzymes [211]. Recent advancements in enzyme engineering have been successfully integrated with immobilization techniques, enabling the development of more stable and active biocatalysts. Strain-promoted azide-alkyne cycloaddition (SPAAC) and artificial amino acid incorporation methods enhance enzyme performance, as demonstrated with lipase and amine transaminases (ATAs) [212; 213].