RESUMEN
Microbial biosensors have diverse applications in metabolic engineering and medicine. Specific and accurate quantification of chemical concentrations allows for adaptive regulation of enzymatic pathways and temporally precise expression of diagnostic reporters. Although biosensors should differentiate structurally similar ligands with distinct biological functions, such specific sensors are rarely found in nature and challenging to create. Using E. coli Nissle 1917, a generally regarded as safe microbe, we characterized two biosensor systems that promiscuously recognize aromatic amino acids or neurochemicals. To improve the sensors' selectivity and sensitivity, we applied rational protein engineering by identifying and mutagenizing amino acid residues and successfully demonstrated the ligand-specific biosensors for phenylalanine, tyrosine, phenylethylamine, and tyramine. Additionally, our approach revealed insights into the uncharacterized structure of the FeaR regulator, including critical residues in ligand binding. These results lay the groundwork for developing kinetically adaptive microbes for diverse applications. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Aminoácidos Aromáticos , Técnicas Biosensibles , Técnicas Biosensibles/métodos , Escherichia coli , Ligandos , FenilalaninaRESUMEN
Enzyme immobilization is a well-known method for the improvement of enzyme reusability and stability. To achieve very high effectiveness of the enzyme immobilization, not only does the method of attachment need to be optimized, but the appropriate support must be chosen. The essential necessities addressed to the support applied for enzyme immobilization can be focused on the material features as well as on the stability and resistances in certain conditions. Ceramic membranes and nanoparticles are the most widespread supports for enzyme immobilization. Hence, the immobilization of enzymes on ceramic membrane and nanoparticles are summarized and discussed. The important properties of the supports are particle size, pore structure, active surface area, volume to surface ratio, type and number of reactive available groups, as well as thermal, mechanical, and chemical stability. The modifiers and the crosslinkers are crucial to the enzyme loading amount, the chemical and physical stability, and the reusability and catalytical activity of the immobilized enzymes. Therefore, the chemical and physical methods of modification of ceramic materials are presented. The most popular and used modifiers (e.g. APTES, CPTES, VTES) as well as activating agents (GA, gelatin, EDC and/or NHS) applied to the grafting process are discussed. Moreover, functional groups of enzymes are presented and discussed since they play important roles in the enzyme immobilization via covalent bonding. The enhanced physical, chemical, and catalytical properties of immobilized enzymes are discussed revealing the positive balance between the effectiveness of the immobilization process, preservation of high enzyme activity, its good stability, and relatively low cost.