## 4 Matrixes for Enzyme Immobilization
### Natural Organic Carriers  
Natural biopolymers, such as cellulose, alginate, chitosan, starch, collagen, gelatin, and pectin, are sustainable, biodegradable alternatives to petroleum-based plastics [35; 36]. Abundant, biocompatible, and rich in reactive site functionalizations, these versatile materials are widely used in medical, environmental, pharmaceutical, and food industries, as well as in biofuel/energy applications [37]. Among these, polysaccharides, e.g., cellulose, are particularly ideal for enzyme immobilization due to their non-toxicity, biodegradability, and modifiable surface hydroxyl groups. \(\beta\) (1\(\rightarrow\)4)-linked D-glucose contains hydrophilic/hydrophobic regions and displays adaptability to chemical, enzymatic, or microbiological modifications, enhancing its application as a support [38]. Alginate is a water-soluble natural polymer derived from brown algae [39], and it is composed of \(\alpha\)-L-glutamate and \(\beta\)-D-mannuronate blocks with functional groups (ether, carboxylate, and hydroxyl) that facilitate enzyme binding via cationic cross-linkers [40]. Its versatility allows it to form calcium alginate beads, hydrogels, and alginate-polyacrylamide composites, enhancing enzyme activity and reusability [41]. Due to tissue-like properties, alginate is also valuable in tissue engineering and cell delivery [42]. Figure 2: Classification of carrier materials for enzyme immobilization. Chitosanan, a mucopolysaccharideacharide derived from chitin, contains abundant amine and hydroxyl groups, enabling direct enzyme binding without cross-linking [43]. Chitosan is widely favored for its availability, low toxicity, biodegradability, and biocompatibility, particularly for use in drug delivery systems [44]. For instance, chitosan nanoparticles and cationic polymer networks enhanced the stability, enzymatic activity, and reusability of protease and lipase, respectively, across a broad range of pH and temperatures [45; 46]. Unlike alginate, chitosan reduces enzyme leaching and can be molded into beads, fibers, membranes, and gels [47]. The existence of numerous amine and hydroxyl groups in the chitosan structure enable the effective binding of different enzymes. While its low rigidity poses storage and catalysis challenges, chemical cross-linking can improve its mechanical strength [48]. Widely used in wastewater treatment, biomedicine, agriculture, and food processing, chitosan remains a robust alternative for enzyme immobilization [49]. Collagen, the most abundant protein in vertebrates, is widely used as a biomaterial due its excellent biological properties and functional groups (e.g., -OH, -COOH, CONH\({}_{2}\) and NH\({}_{2}\)) which facilitate chemical reactions [50]. It has been employed in various forms, such as fibers, powders, and membranes, for enzyme immobilization. Among these, powdered collagen offers a larger surface area and greater versatility in bioreactors, making it particularly advantageous. Additionally, Fe\({}^{3+}\)-collagen fibers serve as effective supports for catalase immobilization, retaining significant enzyme activity even after 26 reuse cycles [51]. Gelatin, an affordable and widely available protein derived from collagen, retains biological properties similar to those of collagen [52]. Due to its low cost and biocompatibility, it is commonly used in drug delivery systems and tissue engineering [53]. Its reactive amino groups make it an effective support for enzyme immobilization. When attached to nanofiber surfaces, gelatin combines its biocompatibility with the nanofiber's mechanical strength. In mixed carrier systems, gelatin enhances the cross-linkage with chromium (III) sulfate or potassium chromium (III) sulfate. Its abundant reactive groups provide strong bonding sites for enzymes. Additionally, gelatin improves enzyme loading efficiency; when paired with calcium alginate [54], it aids calcium phosphate deposition, and in combination with polyester films, it achieves a 75% loading efficiency, surpassing previous results of 50% [55].