Journal of Energy Bioscience 2024, Vol.15, No.5, 314-325 http://bioscipublisher.com/index.php/jeb 316 electron transfer processes essential for biofuel cell operation (Bilal et al., 2017). Additionally, the enzyme's specificity towards the substrate, resistance to inhibitors, and ability to function in the presence of various solvents are important considerations (Singh et al., 2013). The enzyme's natural abundance and cost-effectiveness also play significant roles in the selection process (Zhou et al., 2023). 3.2 Genetic and protein engineering approaches to improve enzyme stability To enhance the stability and performance of enzymes in EBFCs, genetic and protein engineering techniques are employed. These approaches include directed evolution, rational design, and chemical modification. Directed evolution involves iterative rounds of mutagenesis and selection to evolve enzymes with desired traits, such as increased thermal stability or resistance to denaturation (Bilal et al., 2017). Rational design uses computational models to predict and introduce specific mutations that enhance enzyme stability and activity (Singh et al., 2013). Chemical modifications, such as the addition of stabilizing agents or the formation of covalent bonds, can further improve enzyme robustness (Bernal et al., 2018). Combining these techniques can lead to the development of enzymes with superior performance in the challenging environments of biofuel cells (Rehm et al., 2016). 3.3 Enzyme immobilization techniques Enzyme immobilization is a crucial strategy to enhance the operational stability and reusability of enzymes in EBFCs. Various immobilization techniques are employed, including adsorption, covalent bonding, encapsulation, and cross-linking. Adsorption involves the physical attachment of enzymes to support materials, which can be simple but may result in weak binding (Cooney et al., 2008). Covalent bonding creates strong, stable links between enzymes and supports, improving durability and resistance to leaching (Pelosi et al., 2022). Encapsulation traps enzymes within a matrix, protecting them from environmental factors while allowing substrate access (Liu et al., 2021). Cross-linking forms networks of enzyme molecules, enhancing stability and activity (Mateo et al., 2007). Advanced immobilization strategies, such as in situ self-assembly and the use of multifunctional supports, further optimize enzyme performance by ensuring proper orientation and accessibility of active sites (Figure 1) (Rehm et al., 2016; Zhou et al., 2023). Figure 1 Schematic illustration of spider nest-shaped 3D Gox bioanode and Lac biocathode for glucose/oxygen biofuel cells (Adopted from Zhou et al., 2023) 4 Electrode Design and Materials 4.1 Selection of electrode materials The selection of electrode materials is crucial for the performance of enzyme-catalyzed biofuel cells. Various materials have been explored to enhance the efficiency of these cells. Carbon-based materials, such as carbon
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