Glucose Oxidoreductases for Glucose Monitoring: A Bird's Eye View of Glucose-Sensing Enzymes II

Biosensors, utilizing glucose oxidoreductases as biological recognition elements (BREs), underpin current blood glucose monitoring (BGM) and continuous glucose monitoring (CGM) technologies. Glucose oxidase remains the gold standard due to its high selectivity and stability, yet its oxygen dependency limits it to first- and second-generation sensing. Fungal flavin adenine dinucletotide (FAD)-dependent glucose dehydrogenase (GDH) has emerged as the standard for mediator-based second-generation sensors due to its oxygen insensitivity, though it requires further engineering to match the stability and selectivity for CGM applications. Recent research increasingly prioritizes third-generation sensors capable of direct electron transfer (DET). Bacterial FADGDHs are premier candidates, possessing intrinsic DET capabilities via dedicated covalently bound heme subunits, while cellobiose dehydrogenase represents a rising star utilizing non-covalently bound heme b as a built-in mediator. Both require extensive protein engineering to overcome inherent limitations in substrate specificity and catalytic efficiency toward glucose. This review introduces the protein and enzyme sciences of glucose oxidoreductases, which have been and expected to be applied to blood, interstitial fluid, and other biological fluids. It reviews their reported and expected sensing modalities and engineering strategies, including rational design and chimera construction, to enhance enzyme robustness, selectivity, and the availability of electron acceptors, including DET ability. It also introduces perspectives on future engineering approaches to create ideal glucose-sensing oxidoreductases. This review article is an updated version of "Review of Glucose Oxidases and Glucose Dehydrogenases: A Bird's Eye View of Glucose Sensing Enzymes," which was published in 2011 in the Journal of Diabetes Science and Technology (JDST) (https://doi.org/10.1177/193229681100500507).