Wang Feifei,Wang Xinling,Wang Yu,Zhang Yin,Liu Tianxiang,Sun Jian
A novel and facile fluorescence turn-on assay for detecting α-glucosidase (α-Glu) activity and screening inhibitors is reported. This assay exploits α-Glu-catalyzed hydrolysis of L-ascorbic acid-2-O-α-D-glucopyranosyl (AA2G) to release ascorbic acid (AA), triggering an in situ fluorogenic reaction with N-methylethylenediamine (N-MEDA) that generates non-conjugated polymer dots (NCPDs). These NCPDs emit blue fluorescence at 425 nm upon excitation at 350 nm, and the fluorescence intensity increases proportionally with the concentration of α-Glu, enabling quantitative detection of enzymatic activity. The fluorescent assay demonstrates a linear response ranging from 0.01 to 10 U/mL and achieves a low detection limit of 0.0034 U/mL, surpassing most existing methods in sensitivity and width of linear range. In addition, the proposed method has been successfully applied to screen α-Glu inhibitors (AGIs) and determine α-Glu activity in real samples, yielding satisfying results. This work introduces a robust analytical framework and promising approach for clinical diagnosis and anti-diabetic drug discovery. Graphical
Lei Li,Han Lu,Yuling Xue,Yannan He,Baohua Zhao,Lianzhong Ai,Shijie Wang
Probiotic fermented milk has emerged as a prominent functional food for its substantial role in promoting intestinal health and overall wellness. However, a persistent challenge in the industry is post-acidification, a process where lactic acid bacteria continue fermenting during storage, leading to excessive acidity, textural degradation, and flavor alterations. This study focuses on the identification and characterization of a naturally mutated strain of Lacticaseibacillus paracasei capable of significantly attenuating post-acidification in fermented milk. A novel mutation was discovered in the α-glucosidase gene (AF91_03255), wherein the substitution of arginine at position 477 with glycine altered the enzyme's binding dynamics. This mutation enhanced binding stability between the enzyme and its substrate by approximately 16.8%, indirectly modulating acid production without directly limiting glucose metabolism. Through a combination of multi-omics techniques, including metabolomics and genomic sequencing, this study revealed significant shifts in the glycerophospholipid metabolic pathway, suggesting alterations in cell membrane composition as a core mechanism. Additionally, comparative analyses between the wild-type and mutant strains demonstrated notable differences in acid production, viable cell counts, and storage stability. The mutant strain maintained a higher pH and reduced titratable acidity during extended storage while preserving bacterial viability, distinguishing it from traditional approaches reliant on additives or processing modifications. The findings underscore the potential of α-glucosidase as a pivotal target for post-acidification regulation in fermented milk products. Furthermore, this work contributes valuable insights into the strain improvement strategies essential for advancing the stability and sensory qualities of fermented milk.
Enzyme cascades have attracted widespread attention owing to the exceptional specificity and efficient signals transduction, however, constrained by the high cost and limited stability of bio-enzymes. In this study, a novel mimic multienzyme nanoreactor (GOx@MAF-7(Fe), i.e. GMF) was developed through a one-step encapsulation of glucose oxidase (GOx) into a metal azolate framework, MAF-7(Fe). Benefiting from the synergistic effect of GOx and the exceptional peroxidase-like (POD) activity of MAF-7(Fe), GMF enabled a robust cascade catalytic reaction for colorimetric sensing. The unique structural and functional properties of MAF-7(Fe) not only facilitated efficient enzyme immobilization but also enhanced the stability of GOx, outperforming free enzymes in terms of storage and thermal tolerance. The GMF-based platform demonstrated high sensitivity and selectivity in glucose response. More importantly, by integrating α-glucosidase (α-Glu) into a three-enzyme cascade system, a colorimetric assay was successfully developed for α-Glu activity with a detection limit of 0.25 mU/mL, surpassing most existing methods. This platform was further applied for α-Glu inhibitor screening, with acarbose as a model inhibitor, and achieved precise quantification of inhibition efficiency (IC 50 = 60.06 nM). This work not only establishes a versatile and efficient sensing platform for diabetes-related biomolecule detection but also pioneers a novel strategy for enzyme immobilization and multienzyme cascade construction, opening new avenues for multifunctional material design in biomedical research.
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