Journal of Energy Bioscience 2024, Vol.15, No.5, 314-325 http://bioscipublisher.com/index.php/jeb 315 This study aims to provide a comprehensive overview of the design and performance optimization of enzyme-catalyzed biofuel cells (EBFCs). It seeks to explore the fundamental principles of EBFCs, the latest advancements in enzyme immobilization and electrode design, and the challenges that need to be addressed for their commercial viability. The study aims to identify opportunities to enhance the efficiency and stability of EBFCs, thereby paving the way for their broader applications in various fields, including environmental technologies and biomedical devices. 2 Fundamentals of Enzyme-Catalyzed Biofuel Cells 2.1 Basic principles and mechanisms of biofuel cells Enzyme-catalyzed biofuel cells (EBCs) are devices that convert chemical energy into electrical energy using biocatalysts, specifically enzymes, to facilitate redox reactions. These cells operate by oxidizing a fuel substrate at the anode and reducing an oxidant at the cathode, with the enzymes acting as catalysts to enhance the reaction rates. The fundamental mechanism involves the transfer of electrons from the fuel to the electrode through the enzyme, which can occur via direct electron transfer (DET) or mediated electron transfer (MET) (Cooney et al., 2008; Meredith and Minteer, 2012). 2.2 Role of enzymes in biofuel cells Enzymes play a crucial role in EBCs by catalyzing the oxidation and reduction reactions necessary for energy conversion. They provide specificity for the substrates and operate under mild conditions, making them suitable for biofuel cells. Enzymes such as glucose oxidase (GOx) and fructose dehydrogenase (FDH) are commonly used due to their ability to facilitate efficient electron transfer processes (Kamitaka et al., 2007; Christwardana et al., 2018). The stability and activity of these enzymes are critical for the performance and longevity of the biofuel cells (Chung et al., 2016; Christwardana et al., 2017). 2.3 Types of enzyme catalysts used Various enzymes are employed in EBCs, each selected based on the specific fuel and desired reaction. Commonly used enzymes include: Glucose oxidase (GOx): Catalyzes the oxidation of glucose to gluconolactone, often used in conjunction with other enzymes or mediators to enhance performance (Hyun et al., 2015; Christwardana et al., 2017; Christwardana et al., 2018). Fructose dehydrogenase (FDH): Facilitates the oxidation of fructose, often used in DET-type bioelectrocatalysis (Kamitaka et al., 2007; Bollella et al., 2018). Horseradish peroxidase (HRP): Used in combination with GOx to improve the reduction of hydrogen peroxide, enhancing the overall catalytic activity and stability (Chung et al., 2018). Alcohol dehydrogenase (ADH), Aldehyde dehydrogenase (A1DH), and Formate dehydrogenase (FDH): These enzymes work in a cascade to achieve complete oxidation of methanol to carbon dioxide, demonstrating the use of multi-enzyme systems for complex substrates (Kar et al., 2011). 2.4 Electron transfer mechanisms in enzyme-catalyzed biofuel cells Electron transfer in EBCs can occur through two primary mechanisms: Direct electron transfer (DET): In this mechanism, electrons are transferred directly between the enzyme and the electrode without the need for mediators. This is facilitated by the close proximity and proper orientation of the enzyme's active site to the electrode surface. Enzymes like FDH and certain multi-copper oxidases have shown efficient DET capabilities (Kamitaka et al., 2007; Meredith and Minteer, 2012; Bollella et al., 2018). Mediated electron transfer (MET): This involves the use of redox mediators that shuttle electrons between the enzyme and the electrode. Mediators can enhance the electron transfer rate and are often used when DET is inefficient or not feasible. The choice of mediator and its interaction with the enzyme and electrode are critical for optimizing the performance of MET-based biofuel cells (Cooney et al., 2008; Meredith and Minteer, 2012). 3 Enzyme Selection and Engineering 3.1 Criteria for selecting enzymes The selection of appropriate enzymes is a critical step in the design of enzyme-catalyzed biofuel cells (EBFCs). Key criteria include the enzyme's catalytic efficiency, stability under operational conditions, and compatibility with the biofuel cell's design. Enzymes such as oxidoreductases are often preferred due to their ability to facilitate
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