Biomimetic immobilization of α-glucosidase inspired by antibody-antigen specific recognition for catalytic preparation of 4-methylumbelliferone.

Immobilization technology plays an important role in enhancing enzyme stability and environmental adaptability. Despite its rapid development, this technology still encounters many challenges such as enzyme leakage, difficulties in large-scale implementation, and limited reusability. Drawing inspiration from natural paired molecules, this study aimed to establish a method for immobilized α-glucosidase using artificial antibody-antigen interaction. The proposed method consists of three main parts: synthesis of artificial antibodies, synthesis of artificial antigens, and assembly of the artificial antibody-antigen complex. The critical step in this method involves selecting a pair of structurally similar compounds: catechol as a template for preparing artificial antibodies and protocatechualdehyde for modifying the enzyme to create the artificial antigens. By utilizing the same functional groups in these compounds, specific recognition of the antigen by the artificial antibody can be achieved, thereby immobilizing the enzymes. The results demonstrated that the immobilization amount, specific activity, and enzyme activity of the immobilized α-glucosidase were 25.09 ± 0.10 mg/g, 5.71 ± 0.17 U/mgprotein and 143.25 ± 1.71 U/gcarrier, respectively. The immobilized α-glucosidase not only exhibited excellent reusability but also demonstrated remarkable performance in catalyzing the hydrolysis of 4-methylumbelliferyl-α-D-glucopyranoside.

Time-Separated Pulse Release-Activation of an Enzyme from Alginate-Polyethylenimine Hydrogels Using Electrochemically Generated Local pH Changes.

ß-Glucosidase (EC 3.2.1.21) from sweet almond was encapsulated into pH-responsive alginate-polyethylenimine (alginate-PEI) hydrogel. Then, electrochemically controlled cyclic local pH changes resulting from ascorbate oxidation (acidification) and oxygen reduction (basification) were used for the pulsatile release of the enzyme from the composite hydrogel. Activation of the enzyme was controlled by the very same pH changes used for ß-glucosidase release, separating these two processes in time. Importantly, the activity of the enzyme, which had not been released yet, was inhibited due to the buffering effect of PEI present in the gel. Thus, only a portion of the released enzyme was activated. Both enzymatic activity and release were monitored by confocal fluorescence microscopy and regular fluorescent spectroscopy. Namely, commercially available very little or nonfluorescent substrate 4-methylumbelliferyl-ß-d-glucopyranoside was hydrolyzed by ß-glucosidase to produce a highly fluorescent product 4-methylumbelliferone during the activation phase. At the same time, labeling of the enzyme with rhodamine B isothiocyanate was used for release observation. The proposed work represents an interesting smart release-activation system with potential applications in biomedical field.