## 5 Innovative Approaches for Enzyme Immobilization
### 3D-Printing Technology for Enzyme Immobilization  
3D printing (additive manufacturing) is an advanced fabrication method for creating complex, customizable structures with applications in tissue engineering, industrial design, and beyond [230]. This technology allows for precise control over enzyme loading and distribution, as well as the optimization of immobilization factors such as interconnectivity, surface contact, and porosity [231]. Additionally, it offers cost-effective production, intricate geometries, low material costs, reduced labor, scalability, and minimal waste, while avoiding toxic chemicals [232].  
Extrusion-based printing, fused deposition modeling (FDM), and stereolithography are prominent 3D printing techniques for enzyme immobilization (SLA) [233]. Among these, FDM is widely favored due to its affordability, low material loss, and ease of material replacement [60]. Successful immobilization requires materials with chemical resistance, mechanical stability, non-toxicity, and enzyme compatibility [234].  
The use of 3D printing is advancing enzyme immobilization across medical, biosensing, and industrial applications, including enzyme cascade reactions. For instance, FDM has been used to fabricate scaffolds for immobilizing four distinct enzymes [235], while 3D-printed nanocellulose scaffolds from soybean and rice effectively immobilize sucrose synthase (SuSy) and C-glucosyltransferase(CGT) [236]. By optimizing scaffold properties of enzyme activity, efficiency and reproducibility can be significantly enhanced.  
The integration of 3D printing with biocatalysis offers promising biotechnological applications. For instance, xylanase immobilized on 3D-printed sodium alginate (SA) microspheres efficiently degraded lignocellulosic in corn cobs, demonstrating high catalytic activ  
Figure 7: Two different approaches for cross-linked enzyme aggregates. (**A**) Conventional CLEA formation; (**B**) Polymer-supported CLEA synthesis.  
ity and reusability, providing an ecofriendly alternative to conventional high-temperature acidic methods [237].  
In environmental protection, 3D printing enables the creation of customized devices for removing toxic contaminants from water. Laccase has reportedly been immobilized by 3D printing using PLA scaffolds and an SLA-printed device [238]. The immobilized laccase showed promising potential, stability, and reusability in removing toxic pollutants like estrogen diclofenac and ethinylestradioliol from waste water.  
The use of 3D printing offers rapid, cost-effective bioelectrode production for medical applications. For example a 3D-printed microneedle biosensor enabled pain-free, continuous glucose monitoring in diabetic mice, a showing high correlation to commercial blood glucose meters [239]. Suitable materials, such as thermoplastics (e.g., PLA) and hydrogels (e.g., poly (ethylene glycol) diacrylate), enhance bioelectrode performance by supporting enzyme immobilization while maintaining activity and stability [240; 241]. For instance, a hydrogel-based laccase immobilization system demonstrated remarkable stability and reusability in biodegradation processes [242].