Directed evolution engineering to improve activity of glucose dehydrogenase by increasing pocket hydrophobicity.

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.

Carbon dots derived from Poria cocos polysaccharide as an effective"on-off"fluorescence sensor for chromium(Ⅵ)detection / 药物分析学报

Chromium is a harmful contaminant showing mutagenicity and carcinogenicity.Therefore,detection of chromium requires the development of low-cost and high-sensitivity sensors.Herein,blue-fluorescent carbon quantum dots were synthesized by one-step hydrothermal method from alkali-soluble Poria cocos polysaccharide,which is green source,cheap and easy to obtain,and has no pharmacological ac-tivity due to low water solubility.These carbon quantum dots exhibit good fluorescence stability,water solubility,anti-interference and low cytotoxicity,and can be specifically combined with the detection of Cr(Ⅵ)to form a non-fluorescent complex that causes fluorescence quenching,so they can be used as a label-free nanosensor.High-sensitivity detection of Cr(Ⅵ)was achieved through internal filtering and static quenching effects.The fluorescence quenching degree of carbon dots fluorescent probe showed a good linear relationship with Cr(Ⅵ)concentration in the range of 1-100 μM.The linear equation was F0/F=0.9942+0.01472[Cr(Ⅵ)](R2=0.9922),and the detection limit can be as low as 0.25 μM(S/N=3),which has been successfully applied to Cr(Ⅵ)detection in actual water samples herein.