Reversible Regulating the Substrate Specificity of Enzymes in Microgels by a Phase Transition in Polymer Networks

Abstract

Here, we report a distinct approach for regulating the substrate specificity of enzymes immobilized in microgels by a phase transition in polymer networks. The finding is demonstrated on glucose oxidase that is immobilized in thermoresponsive poly(N-isopropylacrylamide)-based microgels. Laser light scattering and enzymatic oxidation tests indicate that the broadened specificity appears at low temperatures, at which the gel matrix is in the relatively swollen state relative to its state at microgel synthesis temperature; upon heating to the relative higher temperatures, the gel matrix is not able to shrink further that offers a tight space in which the enzyme resides to retain high glucose specificity. It is proposed that polymer phase transition in the gel matrix mainly alter protein gates that control passage of substrates into active sites, making them open or close to a certain extent that enable reversible regulating the substrate specificity. The finding is also observed on bulk gels under a rational design, making it of potential interest in enzymatic biofuel cell applications.

Microgels in Tandem with Enzymes: Tuning Adsorption of a pH‐ and Thermoresponsive Microgel for Improved Design of Enzymatic Biosensors

“…Specific examples of thermoregulation include reversible activation‐deactivation of enzymatic reactions, [ 62,65–67 ] switching from homogeneous to heterogeneous enzymatic catalysis, [ 68 ] self‐regulating systems, [ 69 ] or change in substrate specificity. [ 70 ] Both these aspects will be within the scope of our further research.…”

Glucose‐Responsive Enzymatic Cascade Microreactors in Gas‐Shearing Microfluidics Microcapsules

“…It remains, however, important to note that the structural transformations of alginate hydrogels often occur in physiological environments because of competitive ion‐exchange and interconversion reactions. [ 31 ] By the transition in polymer networks, substrate specificity of enzymes immobilized in hydrogels will be regulated, [ 32 ] which is a double‐edged sword and might limit the use of microcapsules in vivo. Although microcapsules are stabler by ionic crosslinking of alginate and chitosan in this work, it remains an open question whether the use of other covalently crosslinked polymer will allow us to design much more robust microcapsules through gas‐shearing method.…”

Responsive microgels and microgel assemblies in biocatalytic applications

“…At lower temperatures, the mutual interaction between the microgel network and GOx led to the stretching of the enzyme structure with the swelling of the microgel, which was attributed to the broadening of specificity towards similar-sized monosaccharides. 90 However, such stimuli can have significant effects on enzyme functioning, and an increase in hydrophobicity and phase change can affect enzyme operation and the transport of substrate and product. Several parameters, including the swelling degree at the first and after repeated swelling-deswelling cycles, and the gel porosity, must be altered to obtain a pre-defined enzyme activity.…”

“…Several parameters, including the swelling degree at the first and after repeated swelling-deswelling cycles, and the gel porosity, must be altered to obtain a pre-defined enzyme activity. 90,95 T A B L E 1 Important use of stimuli responsive properties of the microgel in biocatalytic applications.…”

“…63 All these beneficial attributes of microgel-enzyme conjugates also have some problems, especially with respect to activity loss due to changes in enzyme conformation resulting from chemical cross-linking (structural F I G U R E 3 Effect of microgel binding on enzyme: (A) reversible control of GOx substrate specificity. Reprinted with permission from Reference [90] Copyright © 2022, American Chemical Society. (B) Self-regulated "breathing" hybrid microgel based on urease reaction and chemical composition of hybrid microgel bound to urease.…”