br Conclusion br Conflicts of interest br Introduction Enzym

Conclusion
Conflicts of interest
Introduction Enzymes are widely applied as biocatalysts in fields such as synthetic chemistry [1], pharmaceuticals [2], environmental treatment [3], and food technology [4] because they can catalyze chemical transformations in a highly selective manner under mild reaction conditions. Adenosine deaminase (ADA), a key enzyme in DDD107498 metabolism, catalyzes the irreversible hydrolysis of adenosine and deoxyadenosine to their respective inosine product and ammonia [5]. ADA is also a well-studied model for catalysis through the binding of numerous inhibitors, including ground-state and very potent transition-state analogs, such as inosine, deoxyinosine, and dideoxyinosine [6]. However, the industrial application of free ADA is limited because its stability and catalytic activity are environment-sensitive [7]. Thus, there is great interest in developing new technologies to improve biocatalyst functionality. Enzyme immobilization has been used to optimize processes for industrial applications because immobilization enhances enzyme stability, facilitates separation from the reaction media, and reduces enzyme consumption [8,9]. Enzyme immobilization can be achieved by either physical adsorption or chemical immobilization (e.g., covalent bonding and cross-linking) [10,11]. In physical adsorption, enzyme conformation is largely preserved because the adsorption is mainly achieved by van der Waals' forces or electrostatic interaction [12,13]; however, such bonding is relatively weak and the enzyme easily detaches from the support during use. In contrast, chemical immobilization by covalent bonding or cross-linking usually provides much stronger bonding, thus offering better stability and reusability than physical adsorption. The cross-linking agent glutaraldehyde (GLU) is used for bonding with amino groups on the protein surface [14]. However, these approaches often lead to conformational changes of natural enzymes, thus reducing enzyme specificity [15]. The fabrication of biocompatible micro- and nanoparticles has attracted widespread interest because of their potential application as tools for enzyme immobilization [16–18]. With the development of mesoporous materials and advances in enzyme technology, this field has quickly expanded, providing opportunities for novel functions and applications. Compared to other metal oxides, TiO2 has unique properties such as high mechanical strength [19], low cost, high physical and chemical stability [20], low toxicity, coordination ability with amine and carboxyl groups [21,22], and good biocompatibility, which make it an ideal candidate for enzyme immobilization [3,23,24]. Hydroxyl groups on the TiO2 surface adsorb enzymes and provide a route for further chemical modification of the particle surface and covalent enzyme-TiO2 bonding, which could improve the immobilization performance [25–27]. Pore size may be the most important parameter; the pores must be wide enough to enable the enzyme to enter and diffuse along the channels, leaving room for new molecules to enter. The use of commercial TiO2 nanoparticles as a support for other types of enzyme immobilization for biosensor preparation has been widely reported [28,29]. The aim of this work was to synthesize mesoporous TiO2 with large pore sizes (20 nm), thus creating new possibilities for immobilization of molecules too large to fit into standard pore sizes while enabling easy surface modification and cross-linking for enzyme cycling stability. We intend to functionalize the TiO2 microparticle supports with amine groups to interact with reactive groups on the enzyme. In this study, we enhanced the stability of biocatalysts by integrating several processes serially, including surface modification of inorganic carriers with the coupling agent 3-aminopropyltriethoxysilane (APTES), GLU cross-linker decoration of the carriers, immobilization, and GLU cross-linking of nanocatalysts.