Glucose dehydrogenase (GDH) is a NAD(P)+ dependent oxidoreductase, which is useful in glucose determination kits, glucose biosensors, cofactor regeneration, and biofuel cells. However, the low efficiency of the catalysis hinders the use of GDH in industrial applications. In this study, an analysis of interactions between eight GDH mutants and NADP+ is powered by AlphaFold2 and Discovery Studio 3.0. The docking results showed that more hydrogen bonds formed between mutants, such as P45A and NADP+, which indicated that these mutants had the potential for high catalytic efficiency. Subsequently, we verified all the mutants by site-directed mutagenesis. It was notable that the enzyme activity of mutant P45A was 1829 U/mg, an improvement of 28-fold compared to wild-type GDH. We predicted the hydrophobicity of the protein-ligand complexes, which was confirmed by an 8-anilino-1-naphthalenesulphonic acid fluorescent probe. The following order of increasing hydrophobicity index was deduced: GDH < N46E < F155Y < P45A, which suggested that the enzyme activity of GDH is positively related to its pocket hydrophobicity. Furthermore, P45A still showed better catalytic ability in organic solvents, reaching 692 U/mg in 10% isopropanol, which was 19-fold that of the wild-type GDH. However, its substrate affinity was affected by organic solvents. This study provides a good theoretical foundation for further improving the catalytic efficiency of GDH.

A glucose biosensor was layer-by-layer assembled on a modified glassy carbon electrode (GCE) from a nanocomposite of NAD(P)+-dependent glucose dehydrogenase, aminated polyethylene glycol (mPEG), carboxylic acid-functionalized multi-wall carbon nanotubes (fMWCNTs), and ionic liquid (IL) composite functional polymers. The electrochemical electrode was denoted as NF/IL/GDH/mPEG-fMWCNTs/GCE. The composite polymer membranes were characterized by cyclic voltammetry, ultraviolet-visible spectrophotometry, electrochemical impedance spectroscopy, scanning electron microscopy, and transmission electron microscopy. The cyclic voltammogram of the modified electrode had a pair of well-defined quasi-reversible redox peaks with a formal potential of −61 mV (vs. Ag/AgCl) at a scan rate of 0.05 V s−1. The heterogeneous electron transfer constant (ks) of GDH on the composite functional polymer-modified GCE was 6.5 s−1. The biosensor could sensitively recognize and detect glucose linearly from 0.8 to 100 µM with a detection limit down to 0.46 μM (S/N = 3) and a sensitivity of 29.1 nA μM−1. The apparent Michaelis–Menten constant (Kmapp) of the modified electrode was 0.21 mM. The constructed electrochemical sensor was compared with the high-performance liquid chromatography method for the determination of glucose in commercially available glucose injections. The results demonstrated that the sensor was highly accurate and could be used for the rapid and quantitative determination of glucose concentration.