Electrical Field Reversibly Modulates Enzyme Kinetics of Hexokinase Entrapped in an Electro-Responsive Hydrogel

Abstract

In this paper, the potential use of electroresponsive poly(acrylic acid) (PAA) gels as reversible enzyme activity regulators is analyzed. This was evaluated by measuring the glucose conversion by hexokinase embedded PAA hydrogels under external electrical stimuli. Hexokinase physically entrapped within PAA gels showed a significant increase in activity under an electrical stimulus as compared to in the absence of a stimulus. Kinetic studies revealed that the change in reaction rate could be attributed to the change of V max under a stimulus, while K m was unaffected by the stimulus, which suggested that the increase in reaction rate under an electrical stimulus was due to increased accessibility of the active site. Optimum stimuli-responsive behavior that resulted in maximum conversion under a stimulus and minimum conversion in the absence of a stimulus was obtained at 5.5 pH and 30 °C. The significant difference between the pH optima for the entrapped enzyme and the pure enzyme can be attributed to the acidic nature of the polymeric matrix. Higher cross-linker concentrations resulted in a reduction of both enzyme release and glucose conversion, and a reasonable trade-off between conversion and release could be obtained at 5% cross-linker concentration. Application of a stepwise electrical stimulus revealed that the entrapped enzymes could sustain responsive properties over multiple cycles of electrical switching. Entrapped hexokinase also showed much better reusability compared to pure hexokinase, a combined result of higher enzyme retention and increased stability. No significant impact of the polymer on the interaction between enzyme and glucose was observed. Thus, this system enables electro-responsive modulation of enzyme activity without any reduction in enzyme activity. The studies revealed that conjugation of electro-responsive polymers to enzymes has the potential to reversibly modulate enzymatic reactions via the application of external electrical stimuli, which is promising for bioprocessing and enzymatic separation applications.

Exploring Emergent Properties in Enzymatic Reaction Networks: Design and Control of Dynamic Functional Systems

“…showed that the accessibility of the substrate toward the active site of hexokinase (HK) entrapped in an electroresponsive hydrogel was altered upon application of an electrochemical potential due to the reversible contraction and expansion of the hydrogel. 104 The reaction kinetics of enzymes can also be modified by trapping enzymatic cascades in a porous conducting metal oxide electrode material. This was done by Morello et al to reversibly recycle a nicotinamide cofactor that was generated with an enzymatic cascade consisting of ferredoxin NADP + reductase, L-malate NADP + oxidoreductase, fumarase, L-aspartate ammonia-lyase, and carbonic anhydrase.…”

Microfluidic fabrication of tunable alginate‐based microfibers for the stable immobilization of enzymes

“…Moreover, there was an absorption peak of amides at 1538 cm -1 in the spectra of the products which was absent in the spectra of PAA, indicating the successful formation of enzyme-PAA conjugates. [60,[63][64][65] It is worth noting that enzyme-alginate conjugates cannot be prepared via EDC/NHS coupling strategy (Supporting Information Figure S4). The enzyme cannot covalently bind to alginate which may be ascribed to the steric hindrance of the cyclic structure to the coupling reaction of COOH in alginate.…”

Chemical Modification for the “Off‐/On” Regulation of Enzyme Activity

“…Lysine Light; temperature; pH Deoxyribonuclease I (DNase I); [1b] peroxidase (HRP); [3a] trypsin, papain, DNase I; [10] lysozyme; [45] glucoamylase (GA), proteinase K (ProK), and horseradish RNase; [46] microperoxidase-11; [47] lipase; [48] chymotrypsin [49] Site-selective covalent modification Tyrosine Temperature, pH, GSH Horseradish peroxidase [ 23a] Noncovalent modification / PEGylation, electrical field 𝛼-amylase; [50] glucose; [51] selenoenzyme; [52] dehydrofolate reductase [53] Creation of a switchable reaction center…”